Detection of protein translocation by beta-galactosidase reporter fragment complementation

ABSTRACT

Methods and compositions are provided for detecting molecular translocations, particularly protein translocations within and between subcellular copartments, using at least two components that exhibit a localization-dependent difference in complementation activity. In particular, alpha-complementing β-galactosidase fragments are provided. These β-galactosidase reporter fragments display significantly enhanced enzymatic activity when one fragment is localized in a membrane. Methods for carrying out no-wash ELISA assays based on the reporter component system are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/572,635, filed May 18, 2004, the entire contents ofwhich are incorporated herein by reference.

TECHNICAL FIELD

This invention relates generally to the field of molecular biology. Morespecifically, the invention provides methods and compositions forenzyme-derived reporter systems for detecting molecular locations and,in particular, detecting protein translocation based on reporter groupconcentration using β-galactosidase.

BACKGROUND OF THE INVENTION

Living cells are exposed to a variety of signals from their micro- andmacro-environment. Signals are detected by receptors present on the cellsurface and are then processed and transduced by biochemical cascadesknown as intracellular signaling pathways. Signal transduction throughintracellular space is a key part of cell communication and response andoften involves the movement—or translocation—of signaling proteins fromone position to another within the cell. Novel methods for monitoringspecific modulation of intracellular pathways in living cells couldprovide new opportunities in drug discovery, functional genomics,toxicology, etc.

Several disease states can be attributed to altered activity ofindividual signaling components, such as protein kinases, proteinphosphatases, transcription factors, etc. These signaling components areattractive as targets for therapeutic intervention. Protein kinases andphosphatases are well described components of several intracellularsignaling pathways. Although the involvement of protein kinases incellular signaling have been studied extensively, detailed knowledge ofsignaling-related translocation events needs convenient technology forits study.

Phosphorylation mediated by protein kinases is balanced by phosphataseactivity. Translocation is observed within the family of phosphatases,and is likely to be indicative of phosphatase activity. See, e.g.,Cossette et al., Exp. Cell Res., 223:459-66 (1996). Protein kinasesoften show a specific intracellular distribution before, during andafter activation and monitoring the translocation processes ofindividual protein kinases or subunits thereof is thus likely to beindicative of their functional activity. Connection betweentranslocation and catalytic activation has been shown for proteinkinases like protein kinase C, cAMP-dependent protein kinase andmitogen-activated-protein kinase Erk-1. See, e.g., Debernardi et al:,Proc. Natl. Acad. Sci. USA, 93:4577-82 (1996); Sano et al., Brain Res.,688:213-18 (1995).

Commonly used methods of detection of intracellular localization ofprotein kinases and phosphatases include immunoprecipitation, Westernblots and immunocytochemistry. Translocation indicative of proteinkinase C (PKC) activation has been monitored using different approachessuch as immunocytochemistry where the localization of individualisoforms are detected following permeabilization of the cells; taggingall PKC isoforms with a fluorescent-label; chemical tagging of PKC withthe fluorophore Cy3 and genetic tagging of PKCα and of PKCγ and PKCε.See, e.g., Khalil et al., Am. Physiolog. Society, 263:C714 (1992);Godson et al., Biochim. et Biophys. Acta, 1313:69-71 (1996); Bastiaenset al., Proc. Natl. Acad. Sci. USA, 93:8407-612 (1996); Wagner et al.,Exp. Cell Res., 258:204-14 (2000); Sakai et al., Soc. Neurosci., 22:371,Abstract 150.1 (1996).

Steroid receptors are hormone-dependent activators of gene expression.Steroid receptors mediate the action of steroid hormones (e.g.,glucocorticoids, estrogens, progestins, testosterone, mineralocorticoidsand 1,25-dihydroxycholecalciferol) in human tissues. After activationwith the cognate ligand, receptors bind to chromatin in the nucleus andmodulate the activity of target cellular genes. It is generally acceptedthat the unliganded glucocorticoid receptor (GR) resides in thecytoplasm, and that hormone activation leads both to nuclearaccumulation and gene activation. See, e.g., GASC ET AL., STEROIDHORMONE RECEPTORS: THEIR INTRACELLULAR LOCALISATION 233-50 (Clark, C.R.,ed. Ellis Horwood Ltd. 1987; Beato, Cell, 56:335-44 (1989);Carson-Jurica et al., Endocr. Rev. 11:201-20 (1990); GRONEMEYER, STEROIDHORMONE ACTION 94-117 (Parker, M. G., ed. Oxford University Press 1993);Tsai et al. Annu. Rev. Biochem. 63:451-86 (1994); Akner et al., J.Steroid Biochem. Mol. Biol. 52:1-16 (1995). However, the mechanismsinvolved in nuclear translocation and targeting of steroid receptors toregulatory sites in chromatin have been poorly understood. GreenFluorescent Protein has been used in an assay for the detection oftranslocation of the glucocorticoid receptor. See, e.g., Carey et al.,Cell Biol., 133:985-96 (1996). Methods involving tagging a proteintarget with a luminophore (such as a fluorescent protein like GFP),expressing the luminophore-fusion protein in stably transfected celllines, and quantifying the target movement in response topharmacological stimuli by imaging is the subject of patents such asU.S. Pat. No. 6,518,021; EP 0986753B1; U.S. Pat. No. 6,172,188, and EP0851874.

Directed protein movement in response to external stimuli is a mechanismemployed by eukaryotic signal transduction pathways. Perhaps one of thebest-studied in vivo signal transduction pathways is the NF-κB pathway,a convergent pathway for a number of different stimuli that impact thecell. Ligand binding and other stimulatory events at the cell surfacetrigger activation of the cascade that results in the eventualtranslocation of NF-κB from the cytoplasm to the nucleus. Proteins thatare resident along a pathway offer a potential therapeutic targetingopportunity. Current technologies to track these events are limited tobiochemical fractionation or fusion to fluorescent proteins.

Proteins have been labeled with fluorescent tags to detect theirlocalization and conformational changes both in vitro and in intactcells. Such labeling is essential both for immunofluorescence and forfluorescence analog cytochemistry, in which the biochemistry andtrafficking of proteins are monitored after microinjection into livingcells. See, e.g., Wang et al., eds. METHODS CELL BIOL. 29 (1989).Traditionally, fluorescence labeling is done by purifying proteins andthen covalently conjugating them to reactive derivatives of organicfluorophores. However, the stoichiometry and locations of dye attachmentare often difficult to control, and careful repurification of theproteins is usually necessary.

Biochemical methods are often the most sensitive and quantitativehowever they are limited by their ability to discern subcellularstructures without contamination from other organelles. In addition, thenumber of manipulations involved in preparing the samples makes thesemethods cumbersome and prone to high variability. The use of fluorescentproteins to track protein movement has positively impacted the scope anddetail with which translocation events can be monitored. However thelarge amounts of protein necessary for efficient imaging make theseexperiments difficult to perform with toxic proteins and thesupra-physiological levels of target protein can affect the quality ofthe data obtained. Further, the cell to cell variation is high, coupledto moderately low signal to noise ratios, making the assays morequalitative than quantitative.

Enzyme fragment complementation with beta-galactosidase (β-gal) wasfirst shown in prokaryotes. See, e.g., Ullman et al., J. Mol. Biol.24:339-43 (1967); Ullman et al., J. Mol. Biol 32:1-13 (1968);Ullmanetal., J. Mol. Biol. 12:918-23 (1965). Assays based on thecomplementation of enzyme fragments fused to interacting proteins thatregenerate enzymatic activity upon dimerization are particularly wellsuited to monitoring inducible protein interactions. Reviewed in Rossiet al., Trends Cell Biol. 10:1 19-22 (2000). These systems haveimportant advantages including low level expression of the testproteins, generation of signal as a direct result of the interaction,and enzymatic amplification. As a result, they are highly sensitive andphysiologically relevant assays. See, e.g., Blakely et al., Nat.Biotechnol. 18:218-22 (2000). Additionally, assays based on enzymecomplementation can be performed in any cell type of interest or indiverse cellular compartments such as the nucleus, secretory vesicles orplasma membrane. The β-galactosidase complementation system of U.S. Pat.No. 6,342,345 and as described in the literature enzymatically amplifiesof the signal and can be used to monitor interactions in live cells inreal-time. See, e.g., Rossi et al., Proc. Natl. Acad. Sci. USA94:8405-10 (1997); Blakely et al., Nat. Biotechnol. 18:218-22 (2000).

Protein translocation is essential for mammalian cells to effectcellular responses, and convey information intracellularly. The use ofGFP fusion proteins to track protein movement has revolutionized theability to gather data regarding these actions and has been particularlyuseful in studying real-time kinetics of protein movement. However thedifficulties associated with quantification of these events, such assmall increases in fluorescence, high cell to cell variability, and thenecessity for high expression levels of the fusion protein, prohibit itsuse in certain applications and limit the data to mainly qualitativemeasurements.

Therefore, what is desired is an assay combining both the localizationaspects of fluorescence or luminescence-based assays, and thesensitivity and quantitative aspects of biochemical assays.

BRIEF SUMMARY OF THE INVENTION

Precise and accurate monitoring of protein translocation permitsscreening for and understanding the interplay key components ofbiologically relevant cellular signaling pathways. Moreover, such assaysprovide a means for screening and identifying modulators of proteintranslocation events that may be useful in the diagnosis, treatment, orprevention of disorders and diseases that can be impacted throughprotein translocation events. Enzymatic assays confer several advantagesin monitoring protein translocation events including signalamplification and a wide variety of substrates for in vivo and in vitrodetection. Provided herein is a novel assay system for monitoringprotein translocation based on enzymatic complementation.

In one aspect, provided herein is a method to assess the localconcentration of a compound, comprising: (a) providing a first reportercomponent, wherein said first reporter component is coupled to a firstcompound of interest; (b) providing a second reporter component capableof forming an active complex with said first reporter component togenerate a detectable signal, wherein said second reporter component issituated at a site of interest; (c) forming said active complex, whereinthe formation results from the association of said first reportercomponent with said second reporter component when both components arepresent at said site of interest; and (d) detecting a signal produced bysaid active complex that is measurably different from the signalgenerated when said compound does not localize to said site of interest,whereby the differences in said signal reflect the local concentrationof said compound at said site of interest. In a specific embodiment, thereporter is a low affinity reporter.

In some embodiments, the compound of interest is a protein orbiologically active fragment thereof.

The site of interest can be within a cell. In other words, in situdetection of protein translocation events can be monitored in real time.In some embodiments, the site of interest is the nucleus, cytoplasm, ormembrane of said cell. Such sites include, but are not limited toendosome, mitochondria, golgi, nuclear membrane, nucleolus, ER, actin ormicrotubule cytoskeleton, lysosome, PML bodies, chromatin, P bodies,plasma membrane (exterior and interior), axon, dendrite, and filopodia.

The association of the first reporter component and the second reportercomponent can be mediated by proximity of the reporter components to oneanother. In some embodiments, the second reporter component is coupledto a second compound of interest. In this case, the association of thefirst reporter component and the second reporter component can bemediated by the binding of the first compound of interest to the secondcompound of interest. Exemplary first and second compounds of interestare a ligand-receptor pair, components of a multimeric receptor, orcomponents of a multimeric protein complex. In some cases, theassociation of the first reporter component and the second receptorcomponent is mediated by the affinity of the first compound of interestto the second compound of interest in the presence of a third compoundof interest. In a particular embodiment, the binding affinity of thefirst compound of interest for second compound of interest is greaterthan the binding affinity of the first and second reporter componentsfor each other. The first and second compounds of interest can beproteins. In some instances, the third compound of interest is aprotein.

Typically, the generation of the detectable signal does not rely on thetranscriptional activation of a reporter construct. For example, theformation of the active complex between the first and second reportercomponents generates a chromogenic, fluorogenic, enzymatic, or otheroptically detectable signal without requiring the transcriptionalactivation of a reporter gene construct. The signal can be detected byany suitable method such as flow cytometric analysis or luminescenceassessment.

In some embodiments, the active complex is an enzymatic complex such as,for example, β-galactosidase. When the enzyme is β-galactosidase, thefirst reporter component is a peptide of β-galactosidase comprisingamino acids 5-51 of β-galactosidase. In some embodiments, the firstreporter component comprises a H31R mutation. Specifically providedherein is a first reporter component is SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:8, or SEQID NO:9. When the enzyme is β-galactosidase, the second reportercomponent can be a fragment of β-galactosidase lacking with at least onemutation or deletion in the region of amino acid 1 to 56. In a specificembodiment, the second reporter component is SEQ ID NO: 7.

The active enzyme complex used in these methods can be any enzymeincluding, but limited to β-lactamase or DHFR. In some embodiments, thefirst and second reporter components are each fluorescent proteins andthe detectable signal results from fluorescence resonance energytransfer (FRET). Luminescent proteins may also be employed. Thus, thefirst reporter component is a luminescent protein and the secondreporter component is a fluorescent proteins and the detectable signalresults from bioluminescence resonance energy transfer (BRET). In someembodiments, the reporters are low-affinity. Any suitable fluorescentprotein can be used including, but not limited to green, red, cyan andyellow fluorescent proteins. In one example, the luminescent protein isa Renilla luciferase or a firefly luciferase.

In some embodiments, the first reporter component, the second reportercomponent, or both can be coupled to the protein of interest as a fusionpolypeptide. The second reporter component can be coupled to a peptidethat localizes in a membrane or an intracellular compartment. Thepeptide can be a triplet SV40 nuclear localization signal.

The first reporter component and second reporter can be provided to thecell by any suitable means. In some embodiments, the reporter componentsare provided in expression vector. The expression vectors can be viralvectors such as retroviral vectors.

The localization of the compound assessed by the methods provided hereincan be inducible, for example, by an intracellular signal cascade. Suchlocalization can be induced in response to a hormone, cytokine,pharmaceutical agent, external stressor, or some combination thereof.

In some embodiments, the localization assessed is the movement of thefirst reporter component away from the site of interest.

In another aspect, provided herein is a method to assess intracellularprotein translocation, comprising: (a) providing a first reportercomponent to a cell, wherein said first reporter component is coupled toa protein of interest; (b) providing a second reporter component capableof forming an active complex with the first low-affinity reportercomponent to generate a detectable signal, to said cell, wherein saidsecond reporter component is localized to a specific subcellular region;(c) forming said active complex, wherein the formation is mediated bythe binding of the first low affinity reporter component to the secondreporter component when both components are localized to said specificsubcellular region; and (d) detecting a signal produced by said activecomplex that is measurably different from the signal generated when saidprotein of interest does not localize to said specific subcellularregion.

In yet another aspect, provided herein is a method for a no-wash ELISAassay for detecting a compound in a sample, comprising: (a) immobilizinga first low-affinity reporter component and a first agent that bindssaid compound on a support; (b) contacting said support with a solutioncomprising said compound; (c) adding a second low affinity receptorcomponent coupled to a second agent that binds said compound; (d)forming an active complex of said first and second reporter components,wherein said complex is mediated by binding of said second agent to saidcompound bound to said first agent; and (e) detecting a signal that ismeasurably different from a signal generated when said compound is notbound by said first or second agent.

In some embodiments, the second agent is an antibody or biologicallyactive fragment thereof specific for the compound. Sometimes, the firstagent is also an antibody or biologically active fragment thereof.

Typically, the formation of the active complex between the first andsecond reporter components generates a chromogenic, fluorogenic,enzymatic, or other optically detectable signal. Sometimes, the activecomplex is an enzymatic complex. For example, the active complex can beβ-galactosidase. When the enzyme is β-galactosidase, the first reportercomponent can be a fragment of β-galactosidase lacking with at least onemutation or deletion in the region of amino acid 11 to 44. In a specificembodiment, the first reporter component is SEQ ID NO: 7. When theactive complex is β-galactosidase, the second reporter component can bea peptide of β-galactosidase comprising amino acids 5-51 ofβ-galactosidase. In some embodiments, the second reporter componentcomprises a H31R mutation. Specifically provided herein is a secondreporter component is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, or SEQ ID NO:6. Alternatively, the α peptide may be the firstreporter component and the ω peptide the second reporter component.

Any suitable support can be employed in this method. For example, thesupport can be glass, silica, plastic, nylon or nitrocellulose. In aspecific example, the support is ELISA plate, bead or particle.

Sometimes, the compound is a protein. In a specific embodiment, thecompound is vitellogenin. In some embodiments, the compound is apollutant. Exemplary pollutants include PCB, flucythrinate, andorganochlorine compounds.

In one aspect, provided herein is a no-wash ELISA kit to detect thepresence of a compound comprising: (a) a first reporter component; (b) afirst agent that specifically binds said compound; (c) a second agentthat specifically binds said compound; (d) a support; and (e)optionally, instructions for use. In some embodiments, the reportercomponents are low affinity.

In another aspect, provided herein is a nucleic acid sequence comprisingSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO:9, a vector comprising thenucleic acid sequence, and a cell comprising the nucleic acid sequenceor the vector. Also provided herein is a polypeptide encoded by thenucleic acid sequences provided herein. Specifically, provided herein isa polypeptide comprising the polypeptide encoded by SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO:8, or SEQ ID NO:9.

In yet another aspect, provided herein is a kit for assessing the localconcentration of a compound comprising: (a) a nucleic acid sequence asset forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO:9, or vectorcomprising said sequence; (b) a nucleic acid sequence as set for in SEQID NO: 7 or vector comprising said sequence; and (c) optionally,instructions for use of said nucleic acid sequences.

In another aspect, provided herein is a method for identifying amodulator of protein translocation, comprising: (a) providing a firstreporter component to a cell, wherein said first reporter component iscoupled to a protein of interest; (b) providing a second reportercomponent capable of forming an active complex with said first reportercomponent to generate a detectable signal, to said cell, wherein saidsecond reporter component is localized to a specific subcellular region;(c) providing a signal to said cell that induces the translocation ofone of said reporter components, wherein the translocation results inthe formation of said active complex via the binding of said firstreporter component to said second reporter component when bothcomponents are localized to said specific subcellular region; (d)contacting said cell with a candidate modulator compound; and (d)detecting a signal produced by said active complex in the presence ofsaid candidate compound relative to that signal produced by said activecomplex in the absence of said candidate modulator compound, wherebysaid candidate compound is identified as said modulator compound whosepresence results in a measurably different signal from the signalgenerated in the absence of said candidate modulator compound.

Further provided herein is a method of sorting or for detecting at leastone live cell where protein translocation is induced or modified in acell mixture, comprising separating the cells of the methods disclosedherein according to the degree they are generate said signal from saidfirst and second reporter components, using flow cytometric cellanalysis.

Also provided herein is a method to visualize intracellulartranslocation in real time comprising detecting the signal generated inthe cells comprising the receptor components disclosed herein usingconfocal microscopy.

In one embodiment, a reporter system is provided for detecting alocation of a molecule on a substrate, the system comprising: a firstlow-affinity reporter component coupled to a putative binding moietylocalized on the substrate; and at least a second low-affinity reportercomponent coupled to the molecule and capable of forming an activecomplex with the first low-affinity reporter component to generate adetectable signal, wherein the formation of the active complex ismediated by binding of the molecule to the putative binding moiety, andfurther wherein the detectable signal is measurably different from asignal generated when first putative binding moiety is not localized ona substrate.

In another embodiment, a reporter system for detecting a location of amolecule on a substrate, the system comprising: a first low-affinityreporter component and a putative binding moiety immobilized adjacent toeach other on the substrate; and at least a second low-affinity reportercomponent coupled to the molecule and capable of forming an activecomplex with the first low-affinity reporter component to generate adetectable signal, wherein the formation of the active complex ismediated by binding of the molecule to the putative binding moiety, andfurther wherein the detectable signal is measurably different from asignal generated when first putative binding moiety is not localized ona substrate.

The invention provides a method for detecting localization of amolecule, said method comprising: (a) providing a first low-affinityreporter component coupled to a putative binding moiety localized on thesubstrate; (b) providing a second low-affinity reporter componentcoupled to the molecule and capable of forming an active complex withthe first low-affinity reporter component to generate a detectablesignal; (c) forming the active complex mediated by binding of themolecule to the putative binding moiety; and (d) detecting a signal thatis measurably different from a signal generated when first putativebinding moiety is not localized on a substrate. In one embodiment, themethod provides a first low-affinity reporter component and a putativebinding moiety immobilized adjacent to each other on the substrateinstead.

In some embodiments, wherein the first and second low-affinity reportercomponents are each inactive, complementary fragments of an enzyme andthe detectable signal results from complementation to generate anenzymatic activity. The enzyme may be β-galactosidase. In a preferredembodiment, first component is an omega-fragment of β-galactosidase andthe second component is an alpha-peptide of β-galactosidase comprising aH31R mutation. In other embodiments, the enzyme is β-lactamase, or DHFR.

In one embodiment, the first and second low-affinity reporter componentsinteract to provide a detectable signal resulting from a ras-dependentreporter gene derived from a Ras Recruitment System (RRS) or a SosRecruitment System (SRS).

In one embodiment, the first and second low-affinity reporter componentsare each fluorescent proteins and the detectable signal results fromfluorescence resonance energy transfer (FRET). In one embodiment, thefirst reporter component is a luminescent protein and the secondlow-affinity reporter component is a fluorescent proteins and thedetectable signal results from bioluminescence resonance energy transfer(BRET). In some embodiments, the fluorescent protein is selected fromthe group consisting of green, red, cyan and yellow fluorescentproteins; and the luminescent protein is a Renella luciferase or afirefly luciferase.

In some embodiments, the formation of an active complex between thefirst and second reporter components generates a chromogenic,fluorogenic, enzymatic, or other optically detectable signal.

In some embodiments, the molecule is a ligand; the putative bindingmoiety is a receptor; and the substrate is membrane of a cell such as anuclear membrane or a plasma membrane. In some embodiments, the ligandis translocated to the membrane in a signal transduction process, or ahormonal treatment, or in response to cellular stress. In someembodiments, the location of the ligand is detected by flow cytometry.

The invention also provides a method for no-wash ELISA assay fordetecting an antigen, the method comprising: (a) immobilizing a firstlow-affinity reporter component and a first antibody to a first epitopeof the antigen adjacent to each other on a microwell plate; (b)contacting the microwell with a sample suspected of containing theantigen; (c) adding a second low-affinity reporter component coupled toa second antibody to a second epitope on the antigen; (d) forming anactive complex mediated by binding of the first and second antibodies tothe antigen; and (e) detecting a signal that is measurably differentfrom a signal generated when the antigen is not bound to the firstantibody on the microwell.

One embodiment of the invention provides a method for no-wash ELISAassay for detecting a pollutant in a tissue sample, the methodcomprising: (a) immobilizing a first low-affinity reporter component andthe tissue sample adjacent to each other on a microwell plate; (b)contacting the microwell with a solution comprising an antibody to thepollutant antigen wherein a second low-affinity reporter component iscoupled to the antibody; (d) forming an active complex of the first andsecond low-affinity reporter components mediated by binding of theantibody to the pollutant antigen; and (e) detecting a signal that ismeasurably different from a signal generated when the pollutant antigenis not bound to the antibody on the microwell. The pollutant may beselected from the group consisting of PCB, flucythrinate andorganochlorine compounds; or the pollutant is detected by assayingvitellogenin as the antigen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically depicts a design of the minimal fragment. In FIG.1A, the right panel shows the first 137 amino acids of β-galactosidasefused to the C-terminus of GFP and serially truncated. These constructswere co-expressed with the ω in C2C12 cells and assayed forβ-galactosidase The left panel of FIG. 1A shows amino acids 1-51 ofβ-galactosidase fused to the N-terminus of GFP, serially truncated, thenco-expressed with the ω fragment. The minimal fragment was defined bycombining the last C (47R) and N (5D) terminal truncations then fused tothe N and C terminus of GFP. These fusions were co-expressed with the ωand assayed for enzyme activity.

FIG. 2 depicts enzyme complementation used to assay nucleartranslocation.

FIG. 2A illustrates the design of the nuclear translocation assay. Inthe left panel, the ω fragment is localized to the nucleus with anuclear localization signal (NLS) and the cytosolic protein of interestis fused to the minimal α peptide. Upon stimulation, the α fusion movesto the nucleus and complements the ω, increasing β-galactosidaseactivity. In the right panel, the ω fragment is tethered to the plasmamembrane using the extracellular and transmembrane regions of EGFR. Thecytosolic α-fusion complements spontaneously until stimulation when ittranslocates to the nucleus which results in a loss of enzyme activity.

FIG. 2B shows a nuclear translocation test system comprising a fusionprotein consisting of the mitogen activated protein kinase kinase (MAPKKor MEK) segment that acts as a nuclear export signal (NES) fused to GFP,the a peptide and a triplet SV40 NLS. At steady state the protein iscytosolic, left panel. In the presence of leptomycin B (right panel) theprotein becomes nuclear.

FIG. 2C shows a gain of signal assay in the left panel. TheNES-GFP-α-NLS protein was coexpressed with the ω-NLS and addition ofleptomycin B for 2.5 hours results in a gain of β-galactosidaseactivity. A loss of signal assay is shown in the right panel.Transduction of cells expressing the tEGFR-ω fusion with theNES-GFP-α-NLS construct results in a loss of β-galactosidase activity inthe presence of leptomycin B.

FIG. 3 shows a nuclear translocation of the glucocorticoid receptormonitored by enzyme complementation.

FIG. 3A shows a time course of cells expressing the GR-α fusion andeither the ω-NLS (Left) or tEGFR-ω (right) were assayed forβ-galactosidase activity after treatment with 1 μM dexamethasone for theindicated times.

FIG. 3B shows dose versus time with cell lines shown in FIG. 3A treatedwith varying concentrations of dexamethasone and assayed over time.

FIG. 3C shows protein localization monitored by flow cytometry. The GR-αcell lines were treated with 1 μM dexamethasone (red) for three hours,then stained with the fluorescent β-galactosidase DDAO substrate andanalyzed by flow cytometry. Following dexamethasone treatment, thetreated cells (red) demonstrated staining for the fluorescentβ-galactosidase substrate.

FIG. 4 shows mutation of the α-peptide to generate weakly complementingmutants.

FIG. 4A shows the crystal structure of wild type β-galactosidase withthe α peptide pictured in yellow (light colored) and the mutationsindicated.

FIG. 4B shows mutations made in the α-peptide fused to the C-terminus ofGFP. These constructs were transduced into cells expressing the tEGFR-ωand assayed for β-galactosidase activity.

FIG. 4C shows a schematic of the membrane translocation assay whereinthe ω fragment is tethered to the plasma membrane using theextracellular and transmembrane regions of the EGFR. Four of the mutantswere fused to the C1A domain of PKCγ and coexpressed with the tEGRF.Their responses to 1 μM PMA for 20 minutes are expressed as a foldinduction over background.

FIG. 4D shows the H31R mutant that showed the highest fold induction,treated with varying levels of PMA and then assayed for β-galactosidaseactivity.

FIG. 4E shows single cell analysis by flow cytometry in live cells usingthe DDAO fluorescent substrate.

FIG. 5 shows translocation of the AKT PH domain monitored byβ-galactosidase complementation.

FIG. 5A shows the AKT-GFP-a fusion protein transduced into 3T3 cellsexpressing the tEGFR-ω fusion. The cells were treated with 50 ng/mlPDGF, insulin, or sorbitol for the indicated times and assayed forβ-galactosidase activity using the luminescent substrate.

FIG. 5B shows sequential stimulation of cells used in FIG. 5A withinsulin, PDGF and Sorbitol followed by assay for β-galactosidaseactivity.

FIG. 6 shows total cell lysates from the cell lines expressing theindicated PH domains fused to the GFP-α chimera used in FIG. 5immunoblotted for expression of the fusion protein. A titration ofrecombinant GFP was included to quantitate the levels of protein.

FIG. 7 shows a schematic of an ELISA assay according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Control of protein localization represents one of the fundamentalmechanisms employed by cells to manage protein-protein interaction andto precisely generate cellular signals. Alterations in proteinlocalization results in modification of basic cellular activationpathways. For example, a change in the localization of a specificprotein may induce or abrogate the activation of the cellular activationpathway involving that protein. Current technologies to monitor proteinlocalization are currently limited to biochemical fractionation orfusion to fluorescent proteins. See, e.g., Lever et al., (1980) andTsien et al., (1998). While some of the biochemical methods demonstrategood sensitivity, these methods are limited by their ability to discernonly a limited number of subcellular structures without contaminationfrom other organelles. Moreover, the number of manipulations involved inpreparing these samples makes these methods cumbersome and introduceshigh variability. Fluorescent proteins increased the scope and detailfor monitoring protein translocation, but remains limited by the largequantity of proteins necessary for efficient imaging. See, e.g., Ballaet al., (2002). The quantity requirement can diminish the quality of thedata, reduces the method's sensitivity, and limits the ability toperform such experiments with toxic proteins. The high cell-to-cellvariation coupled to a moderately low signal to noise ratio rendersthese methods more qualitative than quantitative. Thus, an assay thatcombines the localization aspects of fluorescent proteins such as GFPwith the sensitivity of biochemical assays increases the quantitativepower and sensitivity of the assay without a significant proteinquantity requirement.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications and sequences from GenBankand other databases referred to herein are incorporated by reference intheir entirety. If a definition set forth in this section is contrary toor otherwise inconsistent with a definition set forth in applications,published applications and other publications and sequences from GenBankand other data bases that are herein incorporated by reference, thedefinition set forth in this section prevails over the definition thatis incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

The term “active complex” refers to an enzyme or other protein that iscapable of generating a detectable signal in the absence oftranscriptional activation of a reporter construct. Typically, activecomplexes are enzymes. Such enzymes are capable of catalyzing theproduction of a detectable product directly or indirectly and may bemodified by recombinant techniques to improve their detectable productproduction.

As used herein, the term “antibody” refers to any form of antibody orfragment thereof that specifically binds then antigen of interest. Thus,it is used in the broadest sense and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they specifically bind the target ofinterest. Any specific antibody can be used in the methods andcompositions provided herein. Thus, the term “antibody” encompasses amolecule comprising at least one variable region from a light chainimmunoglobulin molecule and at least one variable region from a heavychain molecule that in combination form a specific binding site for thetarget antigen. As used herein, the term “specific” refers to theselective binding of the antibody to the target antigen epitope suchthat it does not bind irrelevant or unrelated epitopes.

As used herein, a “detectable signal” refers to any detectable signalwhich occurs upon the association of the reporter components or via theinteraction of the associated components with another substance, e.g., asubstrate. Such detectable signals include, but are not limited to achromogenic, fluorescent, phosphorescent, luminescent, orchemiluminescent signal.

As used herein, the term “cell” refers to any cell enclosed by a plasmaor cell membrane. Preferably, the cell is a mammalian cell. The cell canbe human or nonhuman. The cell can be freshly isolated (i.e., primary)or derived from a short term- or long term-established cell line.Exemplary cells include embryonic, neonatal or adult cells, transformedcells (e.g., spontaneously- or virally-transformed), neoplastic, andmalignant cells. These include, but are not limited to fibroblasts,macrophages, myoblasts, osteoclasts, osteoclasts, hematopoietic cells,neurons, glial cells, primary B- and T-cells, B- and T-cell lines,chondrocytes, keratinocytes, adipocytes, hepatocytes, monocytes,endothelial cells, smooth muscle cells, and pericytes. In someembodiments, the cells have been engineered using recombinant technologyto express one or more exogenous proteins or to “knock out” expressionof one or more endogenous proteins. For example, a mammalian cell linemay be engineered to express (or overexpress) a receptor protein.

As used herein, the term “expression system” refers to any suitableexogenous system for expressing the receptor components in the cell. Forexample, exogenous expression of receptor components by a cell asprovided herein can result from the introduction of the nucleic acidsequences encoding the receptor component or a fusion protein comprisinga receptor component in an expression vector. As used herein, the term“fusion protein” refers to a reporter component that is fused to aheterologous protein or protein fragment using recombinant technology.

The term “reporter component” refers to a member of a complex of two ormore subunits that lacks the ability to generate a detectable signal,but is capable to generating a detectable signal when associated withone or more other members of the active complex. Thus, typically thereceptor components used in a cell complement one another such that atleast one biological activity can be detected.

As used herein, the terms “protein”, “polypeptide”, and “peptide” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. It also maybe modified naturally or by intervention; for example, disulfide bondformation, glycosylation, myristylation, acetylation, alkylation,phosphorylation or dephosphorylation. Also included within thedefinition are polypeptides containing one or more analogs of an aminoacid (including, for example, unnatural amino acids) as well as othermodifications known in the art.

Unless otherwise indicated, the practice of the present invention willemploy conventional techniques of molecular biology, biochemistry,microbiology, recombinant DNA, nucleic acid hybridization, genetics,immunology, embryology and oncology which are within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,MOLECULAR CLONING: A LABORATORY MANUAL 2d Ed (Maniatis et al., eds. ColdSpring Harbor Laboratory Press 1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Ausubel, et al., John Wiley & Sons 1987-current edition).

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow. The examples that follow are included for illustrative purposesonly and are not intended to limit the scope of the invention.

Translocation Detection Methods

The compositions and method provided herein allow for rapid,quantitative, in situ monitoring of protein translocation using aprotein component complementation assay system where two inactivecomponents of a reporter protein exhibit a detectable signal when thecomponents associate with one another, indicating that the protein(s)being monitored are in the same location.

The methods provided herein offer several advantages over eitherfluorescent protein or biochemical assays. First, a wide variety ofsubstrates may be employed for in situ analysis, allowing the methods tobe used in histological, flow cytometry, and high-throughput screeningapplications. Second, the small size of the α peptide reduces artifactsand eliminates limitations resulting from the use of large complementingpeptides in assays previously described. The smaller size peptide ismuch less likely to interfere with normal translocation events.Moreover, because both subunits are necessary for complementation,efficient localization of the ω to a specific cellular location ensuressignal generation solely from this region. The assay can be performed inany cell type using the smaller peptide method, including non-adherentcells that are difficult to image using fluorescent proteins. Anotheradvantage lies in the ability of the complemented enzyme to processmultiple molecules of substrate, amplifying the signal obtained from thetranslocation event. The amplification permits the detection of fewernumbers of molecules than microscopy-based assays, thereby allowing thestudied protein to be expressed at physiologically relevantconcentrations. Such an assay is ideal for toxic proteins or thoselocalized by a binding partner that is not exogenously expressed. Afurther advantage of this amplification is to provide a high signal tonoise ratio. The robust signal coupled to the low sample variability,generates a highly sensitive and quantitative assay for proteintranslocation that is applicable to all cell types in a high-throughputscreening fornat.

In one aspect, provided herein is a method to assess the localconcentration of a compound, comprising: (a) providing a first reportercomponent, wherein said first reporter component is coupled to a firstcompound of interest; (b) providing a second reporter component capableof forming an active complex with said first reporter component togenerate a detectable signal, wherein said second reporter component issituated at a site of interest; (c) forming said active complex, whereinthe formation results from the association of said first reportercomponent with said second reporter component when both components arepresent at said site of interest; and (d) detecting a signal produced bysaid active complex that is measurably different from the signalgenerated when said compound does not localize to said site of interest,whereby the differences in said signal reflect the local concentrationof said compound at said site of interest.

In another aspect, provided herein is a method to assess intracellularprotein translocation, comprising: (a) providing a first low affinityreporter component to a cell, wherein said first reporter component iscoupled to a protein of interest; (b) providing a second low affinityreporter component capable of forming an active complex with the firstlow-affinity reporter component to generate a detectable signal, to saidcell, wherein said second reporter component is localized to a specificsubcellular region; (c) forming said active complex, wherein theformation is mediated by the binding of the first low affinity reportercomponent to the second reporter component when both components arelocalized to said specific subcellular region; and (d) detecting asignal produced by said active complex that is measurably different fromthe signal generated when said protein of interest does not localize tosaid specific subcellular region.

Any suitable reporter can be employed in the claimed methods. In aspecific embodiment, the reporter is a protein. The reporter componentsrepresent a complementary portion of the reporter that alone is inactiveor devoid of signal-emitting activity. However, when the reportercomponents interact with one another, the reporter components interactto generate a detectable signal. The interaction of the complementaryreporter components can be mediated by increased localization at aparticular site, exceeding a critical concentration, multimerization,proximity, and the like.

For example, the fragments of the reporter enzyme are made such thatthere is one large fragment being at least 80%, sometimes at least 90%or 95% of the full length parental enzyme, and a smaller fragment thatencodes less than 20% of the native (i.e., non-fragmented) enzymenecessary to complement the larger fragment to result in detectableenzymatic activity. The larger fragment is of sufficient length to beproperly folded and forms the three dimensional or tertiary structureresembling the native enzyme. In some embodiments, complementation maybe achieved with more than 2 enzyme fragments or may include additionalinteracting proteins, i.e., a third protein or other components thatfacilitate the complementation of the two fragments. The tertiarystructure of the large fragment can be determined in solution in theabsence of the complementing fragment using any suitable method. Suchmethods include 1) binding by antibodies that are specific for thefolded state of the enzyme; 2) ability to bind substrate; or 3) similarpattern of accessibility of amino acids exposed to reactive compounds(e.g., solvent and the like) by comparison with the parental enzyme. Apattern of accessibility means that the amino acids on the outside ofthe protein.react with certain reagents first while the ones on theinside are protected using routine methods.

In one embodiment, the reporter components are low affinity components.In other words, the design of the reporter components reduces affinityof the components for one another such that their interaction can beincreased by increasing the concentration of either fragment. Thus, thelow affinity components provided herein generate detectable signal thatis dependent on the local concentration of each of the fragments. Insome embodiments, the interaction is achieved by coupling a firstreporter component to a protein of interest while the second reportercomponent is coupled to a second protein that interacts with the firstprotein when co-localized with the second protein. The affinity of thecomponents is such that the signal generated when components in solutionis detectably less than or undetectable relative to the signal generatedwhen the components are proximal to one another as a result of atranslocation event. For example, low affinity components are usefulwhen examining translocation events within the same cellular compartmentsuch as that from the cytosol to the plasma membrane. Because the lowaffinity components are unable to mediate full complementation,localized increases in concentration drive the generation of detectableenzymatic activity. For example, the necessary increase in concentrationcan be achieved if one reporter component is localized to the interiorof the plasma membrane and the other is fused ot a cytosolic protein.Coexpression of the low affinity reporter components will typicallyresult in a low level of detectable enzymatic activity. However, if thecytosolic fusion protein is induced to translocate to the plasmamembrane, then a detectable different amount of enzymatic activity willresult because of the greater concentration of the low affinity reportercomponents. While the total cytosolic concentration of reportercomponents is the same, the distribution has changed and results in adetectably different signal resulting from the complementing reportercomponents. Thus, the use of low affinity components permits thedetection of movement of proteins within a subcellular compartment.

Any suitable method can be used to design and generate low affinitymutants, including those disclosed herein. For example, deletions,insertions, or point mutations of the primary enzyme sequence of one orboth fragments such that mixing of these fragments under the appropriateconditions for that enzyme in solution provides less than 30% of theactivity obtained with the parental fragments when mixed in equalamounts in the range of 10⁻³ to 10⁻⁶ M, sometimes about 1, 5, 10, 15,20, or 25% of the enzymatic activity of the native enzyme. Furthermore,increasing the concentration of either of the two fragments by at least4-fold results in a measurable increase in enzyme activity. Low affinityfragments are thus defined relative to high affinity fragments. In oneexample, complementation with high affinity fragments occurs at a 1:1ratio, while complementation with low affinity fragments occurs at aratio greater than 1:1 of the large to small fragment.

In other embodiments, the reporter components are high affinitycomponents. Any suitable method can be used to design and generate lowaffinity mutants, including those disclosed herein. High affinitycomponents are generally two fragments of an enzyme with the fragmentshaving sufficiently high affinity such that they can spontaneously bindto each other and reform a fully functional enzyme or enzyme subunit.Typically, at least 5% of enzymatic activity of the native enzyme isachieved when mixed under appropriate conditions in solution, sometimesabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of theenzymatic activity of the native enzyme. Determination of such activitycan be performed using routine methods with the concentration at whichthe complemented enzyme and parental enzyme are compared typically beingthe same with a range of, e.g., between 10⁻³ to 10⁻⁶ M. High affinitycomponents permits monitoring of the presence, absence, or increase ofthe reporter components as they translocate between subcellularcompartments. For example, if one reporter component is localized to adistinct separable cellular compartment such as the nucleus, then thetranslocation of the second component into the nucleus will result indetectable enzymatic activity, thus permitting the analysis of nucleustranslocation. Accordingly, if the amount of high affinity componentsincreases in the nucleus, then the amount of detectable enzymaticactivity will increase proportionally. Typically, large increases in theamount of activity are detectable up to a 1:1 reporter component ratio.

Suitable reporters include β-galactosidase, DHFR, β-lactamase,ubiquitin, ras-based recruitment systems (RRS and SOS), G-proteinsignaling, green fluorescent protein (GFP), fluorescence resonanceenergy transfer (FRET), bioluminescence resonance energy transfer(BRET), fusion-protein based systems such as yeast two hybrid method,and the like. See, e.g., Remy, et al., Science 283:990-93 (1999); Remy,et al., Proc. Natl. Acad. Sci. USA 96:5394-99 (1999); U.S. Pat. Nos.:6,270,964, 6,294,330 and 6,428,951; Wehrman, et al., Proc. Natl. Acad.Sci. USA 99:3469-74 (2002); U.S. patent application. No. 20030175836;Johnsson, et al., Proc. Natl. Acad. Sci. USA 91:10340-44 (1994); U.S.Pat. Nos.: 5,503,977 and 5,585,245; Aronheim, Methods Mol. Biol.250:251-62 (2004); Maroun et al., Nucleic Acids Res. 27:e4 (1999);Aronheim, Nucleic Acids Res. 25:3373-74 (1997); Ehrhard et al., NatureBiotechnol. 18:1075-79 (2000); Remy, et al., Methods 32:381-88 (2004);Pollok et al., Trends Cell Biol. 9:57-60 (1999); Adams, et al., Nature349:694-97 (1991); Fields, et al., Nature 340:245-46 (1989); Ray P. etal., Proc. Natl. Acad. Sci. USA 99:3105-10 (1999); Xu et al., Proc.Natl. Acad. Sci USA 96:151-56 (1999); Ayoub et al., J. Biol. Chem.277:21522-28 (2002); Paulmurugan et al., Cancer Res. 64:2113-19 (2004).

The reporter component can be coupled to the compound of interest usingany suitable method. Thus, the reporter component and one or morecompounds are generally linked either directly or via a linker.Typically, the linker is covalent. For example, when the reportercomponent and the compound are proteins, methods known in the art forlinking peptides can be employed. In one preferred embodiment, thereporter component and the compound comprise a fusion protein thatincludes the reporter component and the compound being assayed. Thefusion protein is expressed from an encoding nucleic acidintracellularly. This method of expression is particularly advantageousfor analysis of protein translocation.

Any suitable expression system can be used to express the reportercomponents in situ. Transformation may be achieved using viral vectors,calcium phosphate, DEAE-dextran, electroporation, cationic lipidreagents, or any other convenient technique known in the art. The mannerof transformation useful in the present invention are conventional andare exemplified in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al., eds. 2000). Exogenous expression of the receptor componentscan be transient, stable, or some combination thereof. Exogenousexpression can be enhanced or maximized by co-expression with one ormore additional proteins, e.g., HIV rev. Exogenous expression can beachieved using constitutive promoters, e.g., SV40, CMV, and the like,and inducible promoters known in the art. Suitable promoters are thosewhich will function in the cell of interest. In one embodiment, theexpression vector is a retroviral vector.

The level of expression of the receptor component is that required todetect the translocation event. One of ordinary skill in the art candetermine the required level of expression for translocation detectionusing assays routinely employed in the art. Generally, the fusion geneconstructs are expressed at low levels in situ to facilitate themonitoring of intracellular translocation events in the presence of,e.g., endogenous regulators of protein translocation. Low expressionlevels reduces and sometimes eliminated artifacts arising fromoverexpression of such proteins. Low level expression is readilyachieved using appropriate promoters, ribosoine binding sites and otherregulatory,elements in expression systems tailored for the specific cellemployed. For example, fusion gene constructs can be introduced intovectors in which they lie upstream of an antibiotic resistance genewhose translation is regulated by the Encephalomyocarditis virusinternal ribosome entry sequence (IRES), and which contain a mutation inthe splice donor/acceptor sequences upstream of the ATG sequenceresponsible for translational initiation of the fusion gene. This typeof construct results in a lower translation efficiency of the firstcoding sequence in a bicistronic message, but does not affecttranslation of the second (antibiotic resistance) sequence, which issolely dependent on the IRES.

Also provided herein are vectors or plasmids containing a nucleic acidthat encodes for a receptor component or a fusion protein comprising areceptor component. Suitable vectors for use in eukaryotic cells areknown in the art and are commercially available or readily prepared by askilled artisan. Additional vectors can also be found, for example, inCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F. M., et al., eds.2000) and SAMBROOK ET AL., ED. MOLECULAR CLONING: A LABORATORY MANUAL2nd Ed. (1989). In one embodiment, the first reporter component isencoded in a different vector or plasmid than the second reportercomponent. In another embodiment, the first reporter component isencoded in the same vector or plasmid as the second reporter component.The reporter components expression systems (or constructs) may beintroduced into mammalian cells by methods available in the art,including, but not limited to, viral vectors, transformation,co-precipitation, electroporation, neutral or cationic liposome-mediatedtransfer, microinjection or gene gun. Once the nucleic acid isincorporated into a cell as provided herein, the cell can be maintainedunder suitable conditions for expression of the reporter components.Generally, the cells are maintained in a suitable buffer and/or growthmedium or nutrient source for growth of the cells and expression of thegene product(s).

In some embodiments, the methods provided herein utilize one or morereporter components whose natural affinity for the other reportercomponent is modified using molecular biology techniques. Modificationsincludes those made by one or more steps of mutagenesis, truncation,modification, insertion, and the like in the nucleic acid sequence ofthe reporter component. See, e.g., CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Ausebel et al., ed. John Wiley & Sons 2003). For example, suchmodification can be introduced using random mutagenesis, deletion, ordirected mutation based on molecular modeling information. In someembodiments, the affinity of the individual reporter components for eachother are reduced to a level where significant detectable signal is notgenerated unless one of the components is localized on a membrane or asolid support. Methods for generating low affinity reporter componentsinclude point mutations, insertion of foreign sequences, and deletionsof residues either internally or from either terminus. See, e.g., Dunnet al., Protein Eng. 2:283-91 (1988); Poussu et al., Proteins 54:681-92(2004). In other words, there is a detectable difference in the signalgenerated when the two components are brought together in solutionversus when at least one component is initially localized on a membraneor immobilized on a support.

Compounds useful in these methods are those whose biological activityinvolves translocation from location in a cell to another and can bereadily coupled to one or more of the reporter components. In someembodiments, the compound is a protein or biologically active fragmentthereof. Exemplary proteins include proteins in one or more signaltransduction cascades, apoptosis regulation, cell-cycle progression,carcinogenesis, metastasis, transcription and/or its regulation,translation and/or its regulation, proteins that affect cellinteractions, cell adhesion molecules (CAMs), ligand-receptor pairs,proteins that participate in the folding of other proteins, and proteinsinvolved in targeting to particular intracellular compartments, such asthe Golgi apparatus, endoplasmic reticulum, ribosomes, chloroplasts andmitochondria.

Exemplary proteins that affect intracellular translocation eventsinclude, but are not to hormones and cytokines that are involved insignal transduction, such as interferons, chemokines, and hematopoieticgrowth factors. Some examples include lymphotoxin, tumor necrosisfactor, Fas ligand (CD95L) TNF-related apoptosis-inducing ligand(TRAIL), transforming growth factors-α and β (TGF-α and TGF-β),macrophage and granulocyte colony stimulating factors, epidermal growthfactor (EGF), nerve growth factor (NGF), platelet-derived growth factor(PDGF), insulin-like growth factors I and II (IGF-I and IGF-II),endorphins, prostaglandins, neurotransmitters, adrenergic receptors, andcholinergic receptors. Other proteins include intracellular enzymes suchas protein kinases, phosphatases and synthases. One exemplary enzyme isinterleukin-1β converting enzyme (ICE) proteases or caspase 8. Proteinsinvolved in the cell cycle include deoxyribonucleic acid (DNA)polymerases, proliferating cell nuclear antigen, telomerase, cyclins,cyclin dependent kinases, tumor suppressors and phosphatases. Proteinsinvolved in transcription and translation include ribonucleic acid (RNA)polymerases, transcription factors, enhancer-binding proteins andribosomal proteins. Proteins involved in cellular interactions such ascell-to-cell signaling include receptor proteins, and peptide hormonesor their enhancing or inhibitory mimics. Thus, the method providedherein employs one or more components of the ligands (such as thoselisted above) and their respective receptors and related signallingcomponents. Specifically, in some embodiments, first and secondcompounds of interest are a ligand-receptor pair, components of amultimeric receptor, or components of a multimeric protein complex.

Additional interactions that can be studied by the practice of theinvention include interactions involved in cell metabolism and cellstructure. These include, but are not limited to, interactions that areinvolved in energy metabolism or which establish or modify the structureof the membranes, cytoplasm, cytoskeleton, organelles, nuclei, nuclearmatrix or chromosomes of cells. Interactions among constituents of theextracellular matrix, or between constituents of the extracellularmatrix and cells, can also be studied with the methods and compositionsof the invention.

Binding of molecules will depend upon factors in solution such as pH,ionic strength, concentration of components of the assay, andtemperature. In the binding assays using reporter systems describedherein, the binding affinity of the binding moieties should be highenough to permit forced complementation between the reporter subunits.Non-limiting examples of dissociation constants of the binding moietiesin an assay solution, such as a buffered system or cell interior, are onthe order of less than about 10⁻⁸ M, for example, less than about 10⁻⁹M, or optionally, between about 10⁻⁹ to 10⁻¹² M, depending upon theproperties of the particular assay system.

Binding between reporter components or between more than one compound ofinterest can be direct or in the form of a complex with one or moreadditional binding species, such as charged ions or molecules, ligandsor macromolecules. Thus, the presence or absence of functional reportermolecule is detected by the presence or absence of a signal produced bythe functional reporter molecule.

In one embodiment, the functional reporter molecule is an enzyme whoseactivity can be monitored by the appearance of a product of theenzymatically catalyzed reaction (e.g., an increase in detectablesignal) or by disappearance of the enzyme substrate (e.g., a decrease inor quenching of detectable signal). In another embodiment, thefunctional reporter molecule can be detected without addition ofexogenous substrate by measurement of some endogenous property (e.g.,luminescence, chemiluminescence). Exemplary systems that permitdetection of a signal without the addition of exogenous substrateinclude, but are not limited to FRET and BRET fluorescent transferprotein systems.

In embodiments where the functional reporter molecule is an enzyme thatconverts a substrate to a detectable product; the detection steptypically first requires contacting the cell with a substrate for thereporter enzyme. The substrate may be contacted with the lysate usingany convenient protocol. The nature of the particular substratenecessarily depends on the nature of the reporter enzyme which ispresent in the two fragments. For example, the substrate can be one thatis converted by the reporter enzyme into a chromogenic product. Ofinterest in certain embodiments are substrates that are converted by theenzyme into a fluorescent product. The amount of substrate that iscontacted with the lysate may vary, but typically ranges from about 1femtomolar to 10 millimolar.

The substrate conversion can be evaluated in whole cells using methodsknown in the art. Exemplary methods include flow cytometric analysis,luminescent analysis, chemiluminescent analysis, histochemistry,fluorescent microscopy, and the like. The translocation events can bemonitored in real time or following a predetermined incubation timeafter the initiating event. The cell is evaluated for the presence orabsence of detectable signal (or product). The particular detectionprotocol employed varies depending on the nature of the detectablesignal. For example, where the detectable product is a fluorescentproduct, the detection protocol employs the use of a fluorescent lightdetection means, e.g., a fluorescent light scanner, which can scan thelysate for the presence of fluorescent signal. The presence or absenceof detectable signal from the signal producing system, e.g., detectableproduct in the cell, is then used to derive information as to whethertranslocation occurred. The presence or absence of a signal in thelysate is indicative of translocation, depending on the design of thereporter components. The signal can be correlated to the translocationevent in a qualitative or quantitative manner. One also can employ athreshold value, whereby any signal above the threshold value representsinsufficient activity and any signal below the threshold valuerepresents sufficient activity. One also can evaluate the signal in aquantitative or a semi-quantitative manner, in which the amount ofsignal detected is used as a direct indication of the level oftranslocation events. The amount of signal detected may be linear ornon-linear relative to the amount of translocation depending on thesensitivity of the reporter molecule and substrate employed. In oneembodiment, a larger amount of signal indicates a greater amount oftranslocation, such that the amount of signal has a direct relationshipwith the amount of translocation.

The above signal evaluation may be accomplished using any convenientmeans. Thus, the signal may be subjectively evaluated by comparing thesignal to a set of control signals. The evaluation may be done manuallyor using a computing or data processing means that compares the detectedsignal with a set of control values to automatically provide a value forthe cell fusion activity. Quantified interactions can be expressed interms of a concentration of signal molecule, translocation modulatingcompound (as described in the section below), or protein componentrequired for emission of a signal that is 50% of the maximum signal(IC₅₀). Also, quantified interactions can be expressed as a dissociationconstant (K_(d) or K_(i)) using kinetic methods known in the art.

Translocation events have occurred when the signal produced by thefunctional reporter molecule in the system is different after exposureto an translocation initiating event or signal than the signal producedin a system in the absence of exposure to this event or signal. In oneembodiment, a difference in signal is a reduction or elimination ofsignal produced by the functional reporter molecule following theinitiating event or signal as compared to the signal produced by thefunctional reporter molecule in the absence of the event or signal. Inother embodiments, a difference in signal is an increase in the signalproduced by the functional reporter molecule following the initiatingevent or signal as compared to the signal produced by the functionalreporter molecule in the absence of the event or signal.

Any suitable initiating event or signal may be employed in the methodsprovided herein. In one embodiment, translocation is inducible. Inducingsignals include the initiation of one or more signaling cascades inresponse to hormones, cytokines, growth factors, pharmaceutical agents,external stressors, or some combination thereof. Cells can be exposed toone or more signals simultaneously or sequentially. Exemplary signalsinclude but are not limited to PMA, irradiation, osmotic shock, heat,cold, hypoxia, tension, lipids, carbohydrates, metal ion withdrawal,calcium changes, growth factor/serum deprivation as well as thoseinducers discussed above.

In the methods provided herein, the signal generated by enzyme activityis often secondary to the translocation event and thus affects the studyof processes in real time. Using high concentrations of dexamethasone,complete translocation of the glucocorticoid receptor occurs in 10-15min, however the signal measured from β-galactosidase activity althoughdetectable at 15 minutes, continued to increase over the next 180 min.By contrast the kinetics of the plasma membrane system was morepredictable. Thus, the detection of the signal may be somewhat delayedrelative to the translocation event itself.

Perhaps most importantly for applications that may utilize thesemethods, is the ability to accurately reflect levels of input and outputin different systems. Using the glucocorticoid receptor in the nucleartranslocation assay, enzyme complementation accurately described theligand concentration at 10-fold intervals over a 1000-fold range. Evenmore sensitive was the C1A domain in the membrane translocation assay,detecting as little as 0.25 nM PMA (30% increase) and saturating at 800nM PMA (1000% increase). In a more physiologically relevant setting, theenzyme complementation system correctly ordered the stimuli according totheir ability to generate phospholipids, sorbitol>PDGF>insulin throughmonitoring the translocation of the AKT PH domain. See Examples below.Further, significant increases in phospholipid production were alsoapparent when the stimuli were added sequentially.

Thus, the methods provided herein permit the detection of translocationevents that typically are not discernible by microscopy because signalgeneration is dependent on protein contact. Moreover, diffuse cellularstructures or locations that are difficult to visualize usingfluorescent proteins are made accessible for analysis by thistechnology.

In one aspect, provided herein is a method of sorting or for detectingat least one live cell where protein translocation is induced ormodified in a cell mixture, comprising separating the cells of themethods provided herein according to the degree they are generate saidsignal from said first and second reporter components, using flowcytometric cell analysis.

In another aspect, provided herein is a method to visualizeintracellular translocation in real time comprising detecting the signalgenerated in the cells of the methods provided herein using confocalmicroscopy.

Translocation Detection Methods Using β-Galactosidase

In a particular embodiment of the translocation detection assaysproviding herein, the reporter β-galactosidase is employed. The reportercomponents comprise two inactive fragments of β-galactosidase. The firstreporter component is a short α peptide of β-galactosidase. In oneembodiment, the a peptide comprises amino acids 5-51 of β-galactosidase.However, the a peptide can be smaller as long as it includes amino acid46 of β-galactosidase. In some embodiments, the α peptide comprises theH31R mutation. Exemplary α peptides sequences include: (wildtype) SEQ IDNO:1 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQ QL (H31R) SEQ IDNO:2 MGVITDSLAVVLQRRDWENPGVTQLNRLAARPPFASWRNSEEARTDRPSQ QL (F34Y) SEQ IDNO:3 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPYASWRNSEEARTDRPSQ QL (E41Q) SEQ IDNO:4 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSQEARTDRPSQ QL (N39D) SEQ IDNO:5 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRDSEEARTDRPSQ QL (Truncated)SEQ ID NO:6 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRDSEEA

The second reporter component of β-galactosidase is the β-galactosidaseprotein comprising at least one mutation or deletion in the region ofamino acids 1-56, sometimes 11-44. In a specific embodiment, the secondreporter component is the M15 or M112 deletion mutant (ω) oflacZ(β-galactosidase) that is missing amino acids 11-41 or 23-31. SeeLangley et al., Proc. Nat'l Acad. Sci. USA 72:1254-57 (1975). Oneexemplary ω peptide sequence is set forth as SEQ ID NO:7(MGVITDSLAVVARTDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPITQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK).

In one example, the ω peptide is localized to a specific subcellularregion using any suitable method. The ω peptide can be localized to aspecific subcellular region using any suitable method. Exemplary methodsinclude, but are not limited fusion to a targeting peptide, or a peptidesequence that is modified by a cellular process such as lipidmodification, cleavage, or intein modification. The α peptide is fusedto the protein of interest and expressed in the target cell. The ω and αpeptides are typically expressed in silu using an expression system.Typically, each will be expressed in a separate specific subcellularregion. Specific subcellular regions include, but are not limited tonucleus, cytoplasm, membrane, endosome, mitochondria, golgi, nuclearmembrane, nucleolus, ER, actin or microtubule cytoskeleton, lysosome,PML bodies, chromatin, P bodies, plasma membrane (exterior andinterior), axon, dendrite, filopodia, and the like. When the fusionprotein moves into proximity of the ω peptide, the ensuingcomplementation results in enzyme activity that can be monitored in livecells by any suitable method including, but not limited to flowcytometry and a highly sensitive luminescent assay. See, e.g., Nolan etal., Proc. Natl. Acad. Sci. USA 85:2603-07 (1988); Martin et al.,Biotechniques 21:520-24 (1996). Because the signal is amplifiedenzymatically, the translocation events can be detected atphysiologically relevant expression levels of target protein. Thispermits 10-100 fold increase in sensitivity and a 10-foldsignal-to-noise ratio. Further, the enhanced signal generated using thismethod combined with the low sample variability synergize to create ahighly sensitive and quantitative measure of protein localization.

Also provided herein are the nucleic acid sequences as set forth in SEQID NO:1, SEQ ID NO:2. SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, thepolypeptides encoded by these sequences, vectors expressing sequences,as well as cell comprising the vector or the nucleic acid.

In a specific embodiment, the α peptide employed is one that complementsthe ω peptide robustly in mammalian cells. The high affinity minimal apeptide permits sensitive, accurate analysis of protein translocationevents between compartments physically separated by a membrane with onlya minimum of translocation events required for detection. Exemplary highaffinity peptides include SEQ ID NO:8MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQ QL; and (W34Y) SEQ IDNO:9 MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASYRNSEEARTDRPSQ QL.

) Such high affinity a peptides are not suitable for translocationevents that take place in the same compartment such as those from thecytosol to the plasma membrane.

For example, in the embodiment where the chimeric fused protein producedintracellularly includes the alpha peptide and a protein of interest,β-gal activity results when the fused protein moves to the samecompartment as the co peptide and will be proportional to the amount ofprotein that is translocated to that compartment. Thus, the β-galactivity is driven by the translocation of the protein of interest, notby the complementing β-gal peptides themselves. The enzymatic activityserves as readily detectable indicator of that interaction. Anotheradvantage of this system is that only low levels of expression of thetest proteins are required to detect translocation.

Any suitable method for detecting β-gal activity may be employed. Suchmethod include, but are not limited to live-cell flow cytometry andhistochemical staining with the chromogenic substrate5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal). See, e.g.,Nolan et al., Proc. Natl. Acad. Sci. USA 85:2603-07 (1988); LOJDA, Z.,ENZYME HISTOCHEMISTRY: A LABORATORY MANUAL (Springer, Berlin 1979).Histochemical staining for β-gal can be achieved by fixation of cellsfollowed by exposure to X-gal.

In one embodiment, intracellular analyses may be conducted by fixingcells and staining with the indigogenic substrate X-gal. See, e.g.,Mohler and Blau, Proc. Natl. Acad. Sci. U.S.A. 93:12423-27 (1996); U.S.Pat. No. 6,342,345. Fixed cells also can be analyzed by assaying forβ-gal activity by fluorescence histochemistry using an azo dye incombination with either X-gal or 5-bromo-6-chloro-3-indolylβ-D-galactopyranoside (5-6-X-Gal). A preferred combination is the azodye red violet LB (Sigma Chemical, St. Louis, Mo.) and 5-6-X-Gal,referred to as Fluor-X-gal. For this combination, fluorescencemicrographs can be obtained on a fluorescence microscope using arhodamine/Texas Red filter set. Use of these substrates allowsβ-gal-dependent fluorescence to be visualized simultaneously with two ormore other fluorescent signals.

Vital substrates for β-gal, which can be used in living cells, are alsoencompassed by the invention. For example, a vital fluorogenicsubstrate, resorufin β-galactoside bis-aminopropyl polyethylene glycol1900 (RGPEG) has been described. See, e.g., Minden, Bio Techniques20:122-29 (1996). This compound can be delivered to cells bymicroinjection, electroporation or a variety of bulk-loading techniques.Once inside a cell, the substrate is unable to escape through the plasmamembrane or by gap junctions. Another vital substrate that can be usedin the practice of the invention is fluorescein di-β-D-galactopyranoside(FDG), which is especially well-suited for analysis byfluorescence-activated cell sorting (FACS) and flow cytometry. See,e.g., Nolan et al. Proc. Natl. Acad. Sci. USA 85:2603-07 (1988); Rotmanet al., Proc. Natl. Acad. Sci. USA 50:1-6 (1963).

β-gal may also be detected using a chemiluminescence assay. For example,cells containing β-gal fusions are lysed in a mixture of bufferscontaining Galacton Plus substrate from a Galactolight Plus assay kit(Tropix, Bedford Mass.). See, e.g., Bronstein et al, J. Biolumin.Chemilumin., 4:99-111 (1989). After addition of Light EmissionAccelerator solution, luminescence is measured in a luminometer or ascintillation counter.

Representative substrates that are suitable for spectrophotometric orfluorometric analysis include, but are not limited to:p-aminophenyl-β-D-galactopyranoside;2′-N-(hexadecanol)-N-(amino-4′-nitrophenyl)-β-D-galactopyranoside;4-methylumbel-liferyl-β-D-galactopyranoside;napthyl-AS-B1-β-D-galactopyranoside; 1-napthyl-β-D-galactopyranoside;2-napthyl-β-D-galactopyranoside monohydrate;O-nitrophenyl-β-D-galactopyranoside;m-nitrophenyl-β-D-galactopyranoside;p-nitrophenyl-β-D-galactopyranoside; and phenyl-β-D-galacto-pyranoside,5-bromo-4-chloro-3-indolyl-β-D-galactopynanoside,resorufin-β-D-galactopyranoside, 7-hydroxy-4-trifluoromethyl coumarin,Ω-nitrostyryl-β-D-galactopyranoside, andflourescein-β-D-galactopyranoside. See, e.g., U.S. Pat. No. 5,444,161.

Identification of Modulators of Protein Translocation

The methods provided herein can also be employed to identify modulatorsof protein translocation in situ. A method for identifying a modulatorof protein translocation, comprising: (a) providing a first reportercomponent to a cell, wherein said first reporter component is coupled toa protein of interest; (b) providing a second reporter component capableof forming an active complex with said first reporter component togenerate a detectable signal, to said cell, wherein said second reportercomponent is localized to a specific subcellular region; (c) providing asignal to said cell that induces the translocation of one of saidreporter components, wherein the translocation results in the formationof said active complex via the binding of said first reporter componentto said second reporter component when both components are localized tosaid specific subcellular region; (d) contacting said cell with acandidate modulator compound; and (e) detecting a signal produced bysaid active complex in the presence of said candidate compound relativeto that signal produced by said active complex in the absence of saidcandidate modulator compound, whereby said candidate compound isidentified as said modulator compound whose presence results in ameasurably different signal from the signal generated in the absence ofsaid candidate modulator compound.

Using the methods provided above, a candidate compound is identified asa modulator of in situ protein translocation event when the signalproduced by the functional reporter molecule in the system contactedwith the candidate compound is different than the signal produced in asystem not contacted by the candidate compound. A difference in signalcan be a reduction or elimination of signal produced by the functionalreporter molecule in the presence of the candidate compound as comparedto the signal produced by the functional reporter molecule in theabsence of the candidate compound. In other embodiments, a difference insignal can be an increase in signal produced by the functional reportermolecule in the presence of the candidate compound as compared to thesignal produced by the functional reporter molecule in the absence ofthe candidate compound.

A variety of different modulator molecules may be identified using themethod as provided herein. Candidate compounds can encompass numerouschemical classes. In certain embodiments, they are organic molecules,preferably small organic compounds having a molecular weight of morethan 50 and less than about 2,500 daltons. Candidate compounds cancomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and may include at least anamine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups. Candidate compounds can comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate compounds are also include biomoleculeslike peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Candidatecompounds also can include peptide and protein agents, such asantibodies or binding fragments or mimetics thereof, e.g., Fv, F(ab′)₂and Fab.

Candidate compounds also can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

In one embodiment, a candidate compound is identified as a modulator ofprotein translocation when it is capable of specifically modulating thetranslocation of a protein of interest by reducing the rate or amount oftranslocation mediated by the translocation initiating event(s) at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%, often 60, 70, 80 or 90%,and sometimes 100%. In other embodiments, a candidate compound isidentified as a modulator of protein translocation when it is capable ofspecifically modulating the translocation of a protein of interest byincreasing the rate or amount of translocation mediated by thetranslocation initiating event(s) at least about 5, 10, 15, 20, 25, 30,35, 40, 45 or 50%, often 60, 70, 80, 90%, or 100%, and sometime about200, 500, 1000% or greater. As used herein, the term “rate or amount ofprotein translocation” refers to the total quantity of proteintranslocation per unit time. A modulator of protein translocation is onewhose activity results in a change in the cellular response to thetranslocation initiating event. Such cellular responses includeinhibition of apoptosis, inhibition of cell growth, induction ofapoptosis, induction or cell growth, and the like.

The amount of candidate compound that is present in the contact mixturemay vary, particularly depending on the nature of the compound. In oneembodiment, where the agent is a small organic molecule, the amount ofcell fusion inhibitory molecule present in the reaction mixture canrange from about 1 femtomolar to 10 millimolar. In another embodiment,where the agent is an antibody or binding fragment thereof, the amountof the cell fusion inhibitory molecule can range from about 1 femtomolarto 10 millimolar. The amount of any particular agent to include in agiven contact volume can be readily determined empirically using methodsknown to those of skill in the art.

As described above, the presence or absence of detectable signal isdetermined from the translocation of the reporter components into thesame subcellular compartment. The signal can be correlated to themodulatory activity of the candidate and therefore is used to determinethe modulatory activity of a candidate compound using the increase ordecrease of signal as appropriate to the method design. Proteintranslocation modulation can be expressed as % translocation, rate oftranslocation, IC₅₀, K_(d), or other suitable measurement relative tobaseline controls.

The above signal evaluation as a determination of the modulatoryactivity may be accomplished using any convenient means. Thus, thesignal may be subjectively evaluated by comparing the signal to a set ofcontrol signals. The evaluation may be done manually or using acomputing or data processing means.

The above protein translocation protocols are amenable to highthroughput formats, by which is meant that the above translocationassays can be performed in an automated fashion to screen a plurality ofdifferent test cell fusion inhibitor molecules simultaneously. As such,large numbers of compounds can be screened using automated means atsubstantially the same time. In one embodiment, at least about 10,000 to1,000,000 compounds can be screened simultaneously. In these highthroughput formats, one or more of the above steps, including all of thesteps, may be automated, including candidate compound contact, signaldetection and signal evaluation. Such high-throughput assays will beespecially valuable in screening for drugs that influencemedically-relevant protein translocation events.

No-Wash ELISA Method

Provided herein is a method for a no-wash ELISA. Several of theadvantages of the present invention include (1) a single plate can beused to detect more than one analyte, (2) less sample volume is used inthe assay, (3) less reagent volume is used in the assay, (3) the assaycan be adapted to automated protocols, (4) a single plate can be used todetect diseases which have more than one determinant or marker, (5) asingle plate can be used to detect recombinant organisms, includingtransgenic organisms, which express more than one determinant or marker,and (6) a single plate can be used to detect more than one pathogen in asample; (7) there are fewer steps and thus fewer possible technicianerrors; and (8) decreased process time and equipment required foranalysis.

Thus, provided herein is a method for a no-wash ELISA assay fordetecting a compound in a sample, comprising: (a) immobilizing a firstreporter component and a first agent that binds said compound on asupport; (b) contacting said support with a solution comprising saidcompound; (c) adding a second receptor component coupled to a secondagent that binds said compound; (d) forming an active complex of saidfirst and second reporter components, wherein said complex is mediatedby binding of said second agent to said compound bound to said firstagent; and (e) detecting a signal that is measurably different from asignal generated when said compound is not bound. The various steps ofthe assay may be performed sequentially or by combining various steps.For example, steps (b) and (c) may be performed separately orsimultaneously.

In a particular embodiment of the no-wash ELISA assays providing herein,the reporter β-galactosidase is employed. The reporter componentscomprise two inactive fragments of β-galactosidase. One reportercomponent is the M15 deletion mutant (co peptide) oflacZ(β-galactosidase) that is missing amino acids 11-44. The firstreporter can also be the M112 mutant ω peptide. See Langley et al.,Proc. Nat'l Acad. Sci. USA 72:1254-57 (1975). The complementing reportercomponent is a short α peptide of β-galactosidase. The α peptide cancomprise amino acids 5-51 of β-galactosidase. In some embodiments, the αpeptide comprises the H31R mutation. Exemplary α peptides sequencesinclude SEQ ID NO: 1, 2, 3, 4, 5, and 6 as disclosed herein. Thecomplementing reporter component of β-galactosidase is theβ-galactosidase protein comprising at least one mutation or deletion inthe region of amino acids 11-44 or 23-31. One specific ω peptide is asset forth in SEQ ID NO:7. Either component may be immobilized initiallyon the solid support.

Any suitable agent can be used that bind the compound of interest. Inone embodiment, the agent is an antibody. Any suitable antibody orbiologically relevant fragment thereof may be employed in the methodsprovided herein. The antibody of the present methods and compositionscan be monoclonal or polyclonal. The term “monoclonal antibody”, as usedherein, refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic epitope. In contrast, conventional (polyclonal) antibodypreparations typically include a multitude of antibodies directedagainst (or specific for) different epitopes. In one embodiment, thepolyclonal antibody contains monoclonal antibodies with differentepitope specificities, affinities, or avidities within a single antigenthat contains multiple antigenic epitopes. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be. used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clarkson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), forexample.

The antibodies useful in the present methods and compositions can begenerated in cell culture, in phage, or in various animals, includingbut not limited to cows, rabbits, goats, mice, rats, hamsters, guineapigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, theantibody useful in the present methods is a mammalian antibody. Phagetechniques can be used to isolate an initial antibody or to generatevariants with altered specificity or avidity characteristics. Suchtechniques are routine and well known in the art. In one embodiment, theantibody is produced by recombinant means known in the art. For example,a recombinant antibody can be produced by transfecting a host cell witha vector comprising a DNA sequence encoding the antibody. One or morevectors can be used to transfect the DNA sequence expressing at leastone VL and one VH region in the host cell. Exemplary descriptions ofrecombinant means of antibody generation and production include Delves,ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, etal., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993);CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recentedition).

Any form of the antigen can be used to generate the antibody that issufficient to generate a specific antibody for the target antigen. Thus,the eliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents known in the art. The eliciting antigen may be anisolated full-length protein, a cell surface protein (e.g., immunizingwith cells transfected with at least a portion of the antigen), or asoluble protein (e.g., immunizing with only the extracellular domainportion of the protein). The antigen may be produced in a geneticallymodified cell.

Any suitable compound can be detected using these methods. In someembodiments, the compound is an antigen. In one embodiment, the antigenis a pollutant. Exemplary pollutants include, but are not limited toPCB, flucythrinate, and organochlorine compounds. In a specificembodiment, the pollutant is vitellogenin.

In some embodiment, the no-was ELISA method may be used to detect thepresent of antibodies in a sample, typically a biological sample such asblood, serum, urine, and the like.

If a second antibody is required for antigen detection, any suitablemethod can be used to label the antibody. Frequently, the antibodieswill be labeled by joining, either covalently or non-covalently, asubstance which provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. In addition, the antibodies provided herein can be useful asthe antigen-binding component of fluorobodies. See e.g., Zeytun et al.,Nat. Biotechnol. 21:1473-79 (2003).

The reporter component and agents can be immobilized on the support anysuitable means. Exemplary methods include, but are not limited toadsorption, entrapment, and covalent linkages to the support. See, e.g.,CASS ET AL., EDS. IMMOBILIZED BIOMOLECULES IN ANALYSIS: A PRACTICALAPPROACH (Oxford University Press 1998). As used herein, “solid phasesupport” or “solid support”, used interchangeably, is not limited to aspecific type of support. Rather a large number of supports areavailable and are known to one of ordinary skill in the art. Solid phasesupports include silica gels, resins, derivatized plastic films, glassbeads, cotton, plastic beads, and alumina gels. In one embodiment, thesolid support is a 96 well plate, an ELISA plate, bead, particle, glass,silica, plastic, nylon or nitrocellulose.

Suitable substrates and detection methods are described above. Anysuitable means of performing colorimetric, fluorometric, or otheranalysis can be used. The sample may be prepared by any convenientmeans.

Kits

Kits employing the methods described above also are provided herein.Thus, provided herein is a kit for assessing the local concentration ofa compound comprising: (a) a nucleic acid sequence as set forth in SEQID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, or SEQ ID NO:9. or vector comprising said sequence;(b) a nucleic acid sequence as set for in SEQ ID NO:7 or vectorcomprising said sequence; and (c) optionally, instructions for use ofsaid nucleic acid sequences. The compositions provided in the kit mayfurther contain any surfactant, preservative, stabilizer, and enzymeactivator. In some embodiments, standards for calibration of the assayare included.

EXAMPLES Example 1 Design of the Minimal Fragment

α-complementation of β-galactosidase involves a deletion mutant, the ωfragment (ω), that is missing amino acids 11 -41. In E. coli and invitro, it is possible to complement this mutant in trans by providingthe α peptide which encodes amino acids 3-41. However, previous studiesin mammalian cells reported that the smallest complementing fragmentcontained amino acids 1-71. Because a smaller peptide enhances theefficiency of anal sensitivity, the smallest α peptide(s) that wouldcomplement robustly in mammalian cells when fused to an exogenousprotein at the N or C tenninus was determined. The myoblast cell lineC2C12 was engineered to stably express the ω fragment by retroviraltransduction. The α portion of β-galactosidase was fused to theC-terminus of GFP and deletions were made starting at amino acids 137and continuing through amino acids 38. The products were transduced intothe parental cell line expressing the ω-fragment, and the cells weresorted by FACS for similar GFP expression levels.

In FIG. 1A, the right panel shows the first 137 amino acids ofβ-galactosidase fused to the C-terminus of GFP and serially truncated.These constructs were co-expressed with the ω in mouse myoblast C2C 12cells and assayed for β-galactosidase activity using the luminescentassay. Cells were plated in 96-well dishes and assayed forβ-galactosidase activity using a 1,2 dioxetane luminescent substrate. InFIG. 1A, the left panel shows amino acids 1-51 of β-galactosidase fusedto the N-terminus of GFP, serially truncated, then co-expressed with theω fragment. The minimal fragment was defined by combining the lastC-terminal (47R) and N-terminal (5D) truncations which were then fusedto the N and C terminus of GFP. These fusions were co-expressed with theω fragment and assayed for enzyme activity.

Truncations past amino acids 49 result in a 10-fold decrease inβ-galactosidase activity. To determine which residues could be deletedat the N-terminus, amino acids 1-51 were fused to the N-terminus of GFPand deletions were made starting at amino acids 5. These constructs weresimilarly transduced into the parental C2C12 cell line expressing theω-fragment. Deletions past amino acids 5 resulted in a 100-fold decreasein β-galactosidase activity (FIG. 1A left panel), combining thesedeletions the minimal complementing fragment in mammalian cells shouldencompass amino acids 5-49. To ensure this peptide would complement atthe same level when fused to either the amino or carboxy terminus of aprotein, the peptide was fused separately to either end of GFP (FIG.1C). When the peptide was expressed as a C-terminal fusion, robustcomplementation was achieved; however, fusion of the same peptide to theN-terminus resulted in 100 fold less activity. Thus, in order tomaintain consistency for the ensuing studies amino acids 5-51(designated α) is the minimal fragment that complements to high activityin mammalian cells when expressed as a fusion protein in anyorientation.

Example 2 Nuclear Translocation Assay

In order to utilize α complementation as a method of quantitativelyassessing nuclear translocation events, two systems were designed thatare distinct in the localization of the ω-fragment. FIG. 2A is aschematic showing the design of the nuclear translocation assay. In theleft panel, the ω fragment is localized to the nucleus with a nuclearlocalization signal (NLS) and the cytosolic protein of interest is fusedto the minimal α peptide. Upon stimulation the α fusion moves to thenucleus and complements the ω, increasing β-galactosidase activity. Inthe right panel, the co fragment is tethered to the plasma membraneusing the extracellular and transmembrane regions of EGFR. The cytosolicα-fusion complements spontaneously until stimulation when ittranslocates to the nucleus which results in a loss of enzyme activity.

The gain of signal assay (FIG. 2A left panel) localized the ω to thenucleus through fusion to a triplet SV40 nuclear localization signal(NLS). Thus, an increase in β-galactosidase activity should be observedwhen the target protein-α fusion moves from the cytosol to the nucleus.The loss of signal assay (FIG. 2A right panel) localized the ω-fragmentto the plasma membrane through fusion to the extracellular andtransmembrane regions of the EGF receptor (tEGFR). This configurationresulted in a decrease in β-galactosidase activity when the targetprotein-α translocated to the nucleus (FIG. 2A).

As a test system for nuclear translocation, a GFP-α fusion was createdthat contains the nuclear export signal from MEK and a triplet SV40 NLS.See FIG. 2B. Left Panel shows a fusion protein consisting of the mitogenactivated protein kinase kinase (MAPKK or MEK) segment that acted as anuclear export signal fused to GFP, the α peptide and a triplet SV40NLS. See NES; Fisher et al., Cell, 82:475-83 (1995); Wen et al., Cell,82:463-73 (1995). At steady state the protein was cytosolic. In thepresence of leptomycin B, the protein became nuclear.

Thus, at steady-state, the protein was almost exclusively located in thecytosol, but in the presence of leptomycin B, which blocked Crmlmediated nuclear export, the protein was retained in the nucleus. TheGFP-α fusion was transduced separately into C2C12 myoblasts expressingeither the ω-NLS or the tEGFR-ω construct. In the gain of signal assay,addition of leptomycin B for 2 hours resulted in a 3.5 fold increase inβ-galactosidase activity assayed using the luminescent substrate.Similarly, addition of leptomycin B to the loss of signal assay resultedin almost a five-fold decrease in β-galactosidase activity (FIG. 2B,right panel). These results demonstrated that complementation systemscan be used to monitor protein translocation events in mammalian cells.

FIG. 2C shows a gain of signal assay. In the left panel, theNES-GFP-α-NLS protein was coexpressed with the ω-NLS. Addition ofleptomycin B for 2.5 hours resulted in a gain of β-galactosidaseactivity. A loss of signal assay is shown in the right panel.Transduction of cells expressing the tEGFR-w fusion with theNES-GFP-a-NLS construct resulted in a loss of β-galactosidase activityin the presence of leptomycin B.

To test the applicability of the system to a physiological translocationevent the glucocorticoid receptor was fused to the amino-terminus of theGFP-α fusion. The glucocorticoid receptor is a nuclear steroid hormonereceptor that undergoes a robust nuclear translocation in response totreatment with various hormones including dexamethasone. This constructwas stably introduced into both the co-NLS and tEGFR-ω cell lines thenassayed for changes in β-galactosidase activity upon treatment of thecells with dexamethasone using the luminescent β-galactosidasesubstrate.

FIG. 3A shows a time course of cells expressing the GR-α fusion andeither the ω-NLS (Left) or tEGFR-ω (right) and assayed forβ-galactosidase activity after treatment with 1 μM dexamethasone for theindicated times. FIG. 3B shows dose versus time. Cell lines shown inFIG. 3A were treated with varying concentrations of dexamethasone andassayed over time. FIG. 3C shows protein localization monitored by flowcytometry. The GR-α cell lines were treated with 1 μM dexamethasone(red) for 3 hours then stained with the fluorescent β-galactosidase DDAOsubstrate and analyzed by flow cytometry.

Addition of 1 μM dexamethasone for 3 hours to the gain of signal assayresulted in a 3.5 fold induction of β-galactosidase activity, while theloss of signal assay showed a slightly better signal to noise ratio ofapproximately 5-fold (FIG. 3A). The ability to maintain complementationefficiency when fused to the large fusion protein (GR-GFP) suggests thatcomplementation can take place with proteins at least 120 KD in size.

Although differences in β-galactosidase activity were discernible asearly as 30 min of treatment (100% for the gain of signal assay, and 25%for the loss of signal assay) visualization of the translocation eventby GFP fluorescence shows that most of the protein has translocated bythis time, however the differences in β-galactosidase activity continueto accumulate over the next three hours. α complementation in vitrousing purified proteins required 30-60 min to reach equilibrium. The lagin β-galactosidase activity may be due at least in part to the time ofcompetent enzyme formation (gain) and breakdown (loss). Importantly, thedose response assayed over time demonstrated the ability of the systemto discern different levels of stimulus (FIG. 3B). Consistently over aperiod of four hours, the gain of signal assay was able to significantlydetect 10-fold differences in dexamethasone concentration over a 10³fold range. The loss of signal assay showed a higher sensitivity to lowconcentrations of dexamethasone, yet maintained the ability todifferentiate between concentrations ranging from 0.1 to 10 nM.

Assaying protein movement through enzyme complementation permitted theuse of a wide variety of substrates and detection methods, includingseveral fluorescent β-galactosidase substrates. The GR-GFP-α cell lineswere assayed in the presence and absence of dexamethasone for 3 hoursusing the fluorescent β-galactosidase substrate DDAO (FIG. 3C). Bothsystems showed a 5-10 fold change in β-galactosidase activity in thepresence of dexamethasone when assayed in live cells by flow cytometry.Significantly, this was the first report of flow cytometry being used todistinguish cells based solely on the location of an intracellularprotein.

Example 3 α-Peptide Mutants with Diminished Complementation Capacity

The α peptide used in the nuclear translocation assay had the capabilityof spontaneously restoring enzyme activity when placed in the samecellular compartment as the co mutant. Although this method is ideal forproteins that can be separated from the ω by a physical barrier, asystem to identify translocation events within the same cellularcompartment also benefited from α peptides that bind the ω with loweraffinity. β-galactosidase is active only as a tetramer. The crystalstructure of β-galactosidase shows that residues 5-28 of the α peptideare involved in dimerization of two monomers while residues 31-41 areburied within the ω fragment. Without being bound by theory, mutation ofthe buried residues could decrease the ability of the α peptide to dockwith the ω fragment while maintaining its ability to mediate tetramerformation.

Several point mutations spanning residues 31-41 were engineered into theα fused to the carboxy terminus of GFP (FIG. 4). FIG. 4A shows thecrystal structure of wild type β-galactosidase with the α peptidepictured in yellow (light colored) and the mutations indicated. FIG. 4Bshows mutations made in the α-peptide fused to the C-terminus of GFP.These constructs were transduced into the tEGFR-ω cell line, sorted forsimilar amounts of GFP expression, then assayed for β-galactosidaseactivity using the luminescent assay. Of the seven mutations, fivedecreased complemented enzyme activity from 2-25 fold (FIG. 4B).

Example 4 Plasma Membrane Translocation System

Creation of a plasma membrane translocation assay required expression ofthe ω fragment at the membrane which was achieved using the tEGFR-ωconstruct. When the target protein-α fusion move from the cytosol to themembrane, the local increased concentration of α peptide should drivecomplementation as illustrated in FIG. 4B. To determine which of themutants would work best in this type of assay the C1A domain from PKCγwas fused to four of the GFP-α point mutants that complemented tovarying degrees, H31R, E41Q, N39D, and N39Q. FIG. 4C shows a schematicof the membrane translocation assay. The ω fragment was tethered to theplasma membrane using the extracellular and transmembrane regions of theEGFR. Four of the mutants were fused to the C1A domain of PKCγ andcoexpressed with the tEGFR. Their responses to 1 μM PMA for 20 min wereexpressed as a fold induction over background. The C1A domainefficiently translocated to the plasma membrane when exposed to phorbolesters such as PMA (FIG. 4C). The fusion proteins were expressed in NIH3T3 cells expressing the tEGFR-ω, and assayed for increasedcomplementation in the presence of PMA for 30 min. The fold induction ofthe various mutants was inversely proportional to the backgroundactivity for all of the mutants tested as well as the wt peptide whichshowed less than a 10% increase in β-galactosidase activity (FIG. 4A).The H31R mutant, which had the lowest background activity of the mutantstested, showed a remarkable 7-fold induction of β-galactosidase activityin the presence of PMA.

The translocation ofthe C1A domain in response to PMA stimulation hasbeen extensively characterized by fusion to GFP. The largest increase influorescence at the plasma membrane in response to PMA was reported tobe 70% with the lowest dose of PMA detected using these methods beingbetween 10 and 100 nM. Using β-galactosidase complementation, 700-1000%increases in enzyme activity were achieved in the presence of PMA, andas little as 1 nM PMA could be detected in the medium (FIG. 4D). FIG. 4Dshows a H31R dose response. The H31R mutant that showed the highest foldinduction was treated with varying levels of PMA for 30 min and thenassayed for β-galactosidase activity. Like the nuclear translocationassay, single cell analysis by flow cytometry in live cells using theDDAO fluorescent substrate showed a large increase in fluorescence uponinduction of translocation (FIG. 4E). Importantly, the requirement ofthe ω fragment for complementation ensured that enzyme activity was onlygenerated where the ω-fragment was localized. Thus, the assay measuredincreases in protein concentration at a specific location in the cell.

To test whether the system was robust enough to monitor translocationevents in response to physiologically relevant stimuli, this system wasapplied to the translocation of the PH domain of AKT. The AKT PH domainbinds PI 3,4,5 trisphosphate as well as PI 3,4 bisphosphate which aregenerated at the plasma membrane in response to various stimuliincluding peptide growth factors and cellular stresses. The PH domain ofAKT was fused to the GFP-α chimera and expressed in 3T3 fibroblastsharboring the tEGFR-ω protein. FIG. 5A shows the AKT-GFP-α fusionprotein transduced into 3T3 cells expressing the tEGFR-ω fusion. Thecells were stimulated under various conditions, and the translocation ofthe PH domain was assayed in 96-well dishes by a luminescent measure ofβ-galactosidase activity. The cells were treated with 50ng/ml PDGF,insulin, or sorbitol for the indicated times and assayed forβ-galactosidase activity using the luminescent substrate. Osmotic shockshowed the slowest, but largest increase in β-galactosidase activity(8-fold), followed by PDGF (5-fold), and insulin (2.5 fold) which agreedwith biochemical quantifications using radioactive labeling andquantification of phospholipids. However, an advantage of using the PHdomain as a sensor in the β-galactosidase was that only increases at theplasma membrane generate increased in β-galactosidase activity due tothe localization of the ω. Further, the ability to perform these assaysin high-throughput format made it possible to also perform detailed timecourse and dose response experiments.

The high signal to noise ratio and low standard deviations of the enzymecomplementation system combined to generate a highly sensitive andquantitative assay for physiological events. This was most evident whenthe stimuli are applied sequentially. FIG. 5B shows a time course ofstimulation with each of the four stimuli. After each stimulation theβ-galactosidase compleinentation system registered sequential increasesin activity. The cells used in FIG. 5A were sequentially stimulated withinsulin, PDGF and Sorbitol and assayed for β-galactosidase activity.Insulin was applied first, then PDGF, sorbitol. The shapes of each curvewere dependent on the stimulation used, indicating that the rates ofphospholipid generation reflected the magnitude and rate of change inβ-galactosidase activity.

The plasma membrane translocation events may be imaged by using thefluorescence of the GFP molecule fused to the α fragment. However,toxicity associated with stable expression of these domains was aconcern for expressing sufficient protein to image by conventionalconfocal microscopy. The amount of fusion protein expressed wasquantified by immunoblotting the cell lines used in the β-galactosidaseassays with a GFP antibody. The signal was compared to known amounts ofpurified GFP (FIG. 6). The amount of fusion protein expressed per μg wascalculated to be approximately 50 pg/μg of protein for GrpI and 80 pg/μgfor the AKT PH domain or 1 in 12-20,000th of total protein. The C1Adomain was undetectable by Western blot. Assuming a 4 pL volume for 3T3cells the expression level of these domains that were readily assayed byβ-galactosidase activity, was 2-16 nM.

Example 5 Application of the Enzyme Complementation System to No-WashELISA Protocols

The enzyme complementation system to monitor protein localization wasapplicable to a ELISA detection system. (See generally, ELISA and OtherSolid Phase Immunoassays: Theoretical and Practical Aspects, Kemeny etaL, eds. Wiley, John & Sons, Incorporated (1996); Weir's Handbook ofExperimental Immunology, Herzenberg, et al., eds. Blackwell Publishers(1996)). One of the translocations shown to be effectively monitoredusing this technique was movement from the cytosol to the interior ofthe plasma membrane. In the method provided, the large M15β-galactosidase mutant was constitutively localized to the interior of acell membrane. Translocation of a cytosolic protein conjugated to a lowaffinity peptide, either by truncation or mutation, resulted inmeasurable increases in β-galactosidase activity. A significant benefitof using the low affinity components was the elimination washing stepsfrom the ELISA assay.

Using the same concept in in vitro systems using purified proteinsenables a novel form of ELISA detection. An example of this technologyusing a standard Sandwich ELISA is depicted schematically in FIG. 7. Thepurified M15 β-galactosidase mutant low-affinity fragment was coatedonto a substrate along with a primary antibody specific for a particularantigen. Alternately, the low-affinity fragment was conjugated to theantibody and coated on the substrate. The sample containing the antigenwas then added to the plate and consequent binding of the antigen to theprimary antibody occured. A secondary antibody (specific for a differentepitope on the same antigen) conjugated to the complementary lowaffinity fragment (alpha peptide) was then also added. This bindingbrought the secondary antibody and hence the low affinity alpha peptideinto close proximity of the M15 mutant. Because each part of the enzymewas inactive by itself, only the secondary antibody-antigen-primaryantibody complexes produced enzyme (β-gal) activity and thus eliminatedmany of the washing steps involved in standard ELISA assays.

A sandwich ELISA was used as an example but the ELISA can be of anyformat, or in general where proximity of the α-peptide and M15 mutantare used as a readout.

This system could work by proximity but also by immobilization. Thecomplementation of the M15 mutant with the alpha peptide is normally aslow process thus the system may act to immobilize the alpha peptide ina position where it increases its ability to complement. In this casethe complementation is a “presentation” issue. Thus by restraining thealpha peptide to certain configurations in physical space or orientationthat increase its likelihood of binding the M15 mutant increases inenzyme activity can be obtained.

Example 6 ELISA Assay for Detecting Environmental Pollutants

Enzyme-linked immunoassay (ELISA) systems are available for detectingand measuring common environmental pollutants. This is of particularinterest in the light of recent findings of high and potentially harmfullevels of organochlorine compounds, such as PCBs, in some farmed salmon.Assays comprising four different enzyme-linked immunoassay (ELISA)systems for assaying vitellogenin in carp, fathead minnow, medaka, andzebrafish, as well as two enzyme immunoassays (EIAs) for PCB andcoplanar-PCB, are available from Amersham Biosciences (Piscataway,N.J.). An enzyme-linked immunosorbent assay (ELISA) has been developedfor the detection of the insecticide flucythrinate in environmental andfood samples. See, e.g., Nakata et al., Pest Manag. Sci. 57:269-77(2001). Analysis of Soil and Dust Samples for Polychlorinated Biphenylsby Enzyme-linked Immunosorbent Assay also has been reported. See, e.g.,Chuang el al., Analytica Chimica Acta, 376:67-75 (1998).

A purified low-affinity fragment (e.g., M15 β-galactosidase) is coatedonto a substrate such as a microwell plate along with fish cell samplessuspected of containing a pollutant in each well. A primary (ormonoclonal) antibody specific for the particular pollutant (antigen) isconjugated with the complementary low affinity fragment (alpha peptide)is added to each well. Presence of the pollutant in the fish cell sampleis detected by β-galactosidase activity generated by the close proximityof the complementary low affinity fragments. As each complementarylow-affinity fragment of the enzyme is inactive by itself only theformation of the antibody-antigen (pollutant) complexes produces enzyme(β-gal) activity and thus eliminating many of the washing steps involvedin standard ELISA assays.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

1. A method to assess the local concentration of a compound, comprising:(a) providing a first reporter component, wherein said first reportercomponent is coupled to a first compound of interest; (b) providing asecond reporter component capable of forming an active complex with saidfirst reporter component to generate a detectable signal, wherein saidsecond reporter component is situated at a site of interest; (c) formingsaid active complex, wherein the formation results from the associationof said first reporter component with said second reporter componentwhen both components are present at said site of interest; and (d)detecting a signal produced by said active complex that is measurablydifferent from the signal generated when said compound does not localizeto said site of interest, whereby the differences in said signal reflectthe local concentration of said compound at said site of interest. 2.The method of claim 1, wherein the translocation is nuclear and thereporter components are low affinity components.
 3. The method of claim1, wherein the translocation is a plasma membrane translocation and thereporter components are high affinity components.
 4. The method of claim1, wherein said compound of interest is a protein or biologically activefragment thereof.
 5. The method of claim 1, wherein said site ofinterest is within a cell.
 6. The method of claim 5, wherein said siteof interest is the nucleus, cytoplasm, or membrane of said cell.
 7. Themethod of claim 1, wherein the generation of said detectable signal doesnot rely on the transcriptional activation of a reporter construct. 8.The method of claim 1, wherein the association of said first reportercomponent and said second reporter component is mediated by proximity ofsaid reporter components.
 9. The method of claim 1, wherein said secondreporter component is coupled to a second compound of interest.
 10. Themethod of claim 9, wherein the association of said first reportercomponent and said second reporter component is mediated by the bindingof said first compound of interest to said second compound of interest.11. The method of claim 9, wherein the association of said firstreporter component and said second receptor component is mediated by theaffinity of said first compound of interest to said second compound ofinterest in the presence of a third compound of interest.
 12. The methodof claim 9, wherein the binding affinity of said first compound ofinterest for second compound of interest is greater than the bindingaffinity of said first and second reporter components for each other.13. The method of claim 9, wherein said first and second compounds ofinterest are proteins.
 14. The method of claim 11, wherein said thirdcompound of interest is a protein.
 15. The method of claim 1, whereinthe formation of said active complex between said first and secondreporter components generates a chromogenic, fluorogenic, enzymatic, orother optically detectable signal without requiring the transcriptionalactivation of a reporter gene construct.
 16. The method of claim 1,wherein said active complex is an enzymatic complex.
 17. The method ofclaim 1, wherein said active complex is β-galactosidase.
 18. The methodof claim 17, wherein said first reporter component is a peptide ofβ-galactosidase comprising amino acids 5-51 of β-galactosidase.
 19. Themethod of claim 18, wherein said first reporter component comprises aH31R mutation.
 20. The method of claim 17, wherein said first reportercomponent is SEQ ID NO:
 1. 21. The method of claim 17, wherein saidfirst reporter component is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9.
 22. The method ofclaim 17, wherein said second reporter component is a fragment ofβ-galactosidase lacking with at least one mutation or deletion in theregion of amino acid 11 to
 44. 23. The method of claim 17, wherein saidsecond reporter component is SEQ ID NO:7.
 24. The method of claim 16,wherein said active complex β-lactamase or DHFR.
 25. The method of claim1, wherein said first and second reporter components are eachfluorescent proteins and the detectable signal results from fluorescenceresonance energy transfer (FRET).
 26. The method of claim 1, whereinsaid first reporter component is a luminescent protein and said secondreporter component is a fluorescent proteins and the detectable signalresults from bioluminescence resonance energy transfer (BRET).
 27. Thereporter components of claims 25 or 26, wherein said fluorescent proteinis selected from the group consisting of green, red, cyan and yellowfluorescent proteins.
 28. The reporter components of claim 26, whereinsaid first reporter component is a luminescent protein selected from aRenella luciferase or a firefly luciferase.
 29. The method of claim 1,wherein said first reporter component is coupled to said protein ofinterest as a fusion polypeptide.
 30. The method of claim 1, whereinsaid second reporter component is coupled to a peptide that localizes ina membrane or an intracellular compartment.
 31. The method of claim 30,wherein said peptide is a triplet SV40 nuclear localization signal. 32.The method of claim 33, wherein said second reporter component iscoupled to said peptide as a fusion polypeptide.
 33. The method of claim33, wherein said first reporter component is provided to the cell in anexpression vector.
 34. The method of claim 33, wherein said secondreporter component is provided to the cell in an expression vector. 35.The method of claims 33 or 34, wherein said vector is a retroviralvector.
 36. The method of claim 1, wherein said signal is detected byflow cytometry.
 37. The method of claim 1, wherein said signal isdetected by assessing luminescence.
 38. The method of claim 1, whereinthe localization of said compound to said site of interest is inducible.39. The method of claim 38, wherein said localization is induced by anintracellular signal cascade.
 40. The method of claim 38, wherein saidlocalization is induced in response to a hormone, cytokine,pharmaceutical agent, external stressor, or some combination thereof.41. The method of claim 1, wherein the localization assessed is themovement of said first reporter component away from said site ofinterest.
 42. The method of claim 9, wherein said first and secondcompounds of interest are a ligand-receptor pair, components of amultimeric receptor, or components of a multimeric protein complex. 43.A method to assess intracellular protein translocation, comprising: (a)providing a first reporter component to a cell, wherein said firstreporter component is coupled to a protein of interest; (b) providing asecond reporter component capable of forming an active complex with thefirst reporter component to generate a detectable signal, to said cell,wherein said second reporter component is localized to a specificsubcellular region; (c) forming said active complex, wherein theformation is mediated by the binding of the first reporter component tothe second reporter component when both components are localized to saidspecific subcellular region; and (d) detecting a signal produced by saidactive complex that is measurably different from the signal generatedwhen said protein of interest does not localize to said specificsubcellular region.
 44. A method for a no-wash ELISA assay for detectinga compound in a sample, comprising: (a) immobilizing a first reportercomponent and a first agent that binds said compound on a support; (b)contacting said support with a solution comprising said compound; (c)adding a second receptor component coupled to a second agent that bindssaid compound; (d) forming an active complex of said first and secondreporter components, wherein said complex is mediated by binding of saidsecond agent to said compound bound to said first agent; and (e)detecting a signal that is measurably different from a signal generatedwhen said compound is not bound by said first or second agent.
 45. Themethod of claim 44, wherein said second agent is an antibody orbiologically active fragment thereof specific for said compound.
 46. Themethod of claim 45, wherein said first agent is an antibody orbiologically active fragment thereof.
 47. The method of claim 45,wherein the formation of said active complex between said first andsecond reporter components generates a chromogenic, fluorogenic,enzymatic, or other optically detectable signal.
 48. The method of claim44, wherein said active complex is an enzymatic complex.
 49. The methodof claim 48, wherein said active complex is β-galactosidase.
 50. Themethod of claim 49, wherein said second reporter component is a peptideof β-galactosidase comprising amino acids 5-51 of β-galactosidase. 51.The method of claim 49, wherein said second reporter component comprisesa H31R mutation.
 52. The method of claim 49, wherein said secondreporter component is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:6.
 53. The method of claim 49, wherein said firstreporter component is a fragment of β-galactosidase lacking with atleast one mutation or deletion in the region of amino acid 11 to
 44. 54.The method of claim 49, wherein said first reporter component is SEQ IDNO:7.
 55. The method of claim 44, wherein said support is glass, silica,plastic, nylon or nitrocellulose.
 56. The method of claim 44, whereinsaid support is ELISA plate.
 57. The method of claim 44, wherein saidcompound is a protein.
 58. The method of claim 44, wherein said compoundis a pollutant.
 59. The method of claim 58, wherein said pollutant isselected from the group consisting of PCB, flucythrinate, andorganochlorine compounds.
 60. The method of claim 44, wherein saidcompound is vitellogenin.
 61. A no-wash ELISA kit to detect the presenceof a compound comprising: (a) a first reporter component; (b) a firstagent that specifically binds said compound; (c) a second agent thatspecifically binds said compound; (d) a support; and (e) optionally,instructions for use.
 62. A nucleic acid sequence comprising SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:9.
 63. A vector comprising the nucleic acid sequence ofclaim
 62. 64. A cell comprising the nucleic acid sequence of claim 62 orthe vector of claim
 63. 65. A polypeptide encoded by the nucleic acid ofclaim
 62. 66. A polypeptide comprising the polypeptide encoded by SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:9.
 67. A kit for assessing the local concentration of acompound comprising: (a) a nucleic acid sequence as set forth in SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 orSEQ ID NO:9, or vector comprising said sequence; (b) a nucleic acidsequence as set for in SEQ ID NO:7 or vector comprising said sequence;and (c) optionally, instructions for use of said nucleic acid sequences.68. A method for identifying a modulator of protein translocation,comprising: (a) providing a first reporter component to a cell, whereinsaid first reporter component is coupled to a protein of interest; (b)providing a second reporter component capable of forming an activecomplex with said first reporter component to generate a detectablesignal, to said cell, wherein said second reporter component islocalized to a specific subcellular region; (c) providing a signal tosaid cell that induces the translocation of one of said reportercomponents, wherein the translocation results in the formation of saidactive complex via the binding of said first reporter component to saidsecond reporter component when both components are localized to saidspecific subcellular region; (d) contacting said cell with a candidatemodulator compound; and (e) detecting a signal produced by said activecomplex in the presence of said candidate compound relative to thatsignal produced by said active complex in the absence of said candidatemodulator compound, whereby said candidate compound is identified assaid modulator compound whose presence results in a measurably differentsignal from the signal generated in the absence of said candidatemodulator compound.
 69. A method of sorting or for detecting at leastone live cell where protein translocation is induced or modified in acell mixture, comprising separating the cells of claim 1 or 44 accordingto the degree they are generate said signal from said first and secondreporter components, using flow cytometric cell analysis.
 70. A methodto visualize intracellular translocation in real time comprisingdetecting the signal generated in the cells of claim 1 or 44 usingconfocal microscopy.